Isolated donor MHC-derived peptide and uses thereof

ABSTRACT

The invention relates to an peptide derived from a polymorphic region of donor MHC class II molecules which induces tolerance and thus prevents transplant rejection in a patient in need thereof. The invention relates to an isolated peptide of 15 or 16 amino acids long that comprises or consists of the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant for use as drug. The invention relates to an in vitro method for determining whether a transplanted patient is tolerant, comprising a step of determining the presence of CD8+CD45RClow Tregs in a biological sample obtained from said transplanted patient, wherein the presence of CD8+CD45RClow Tregs is indicative of tolerance.

FIELD OF THE INVENTION

The invention is in the field of immunotherapy.

More particularly, the invention relates to an isolated peptide derivedfrom a polymorphic region of donor MHC class II molecules which inducestolerance and thus prevents transplant rejection in a patient in needthereof.

BACKGROUND OF THE INVENTION

Allogeneic human to human transplant remains the best treatment toreplace organs that have failed following disease. The incompatibilitybetween the molecules of the major histocompatibility complex (MHC) ofthe recipient and donor cells is the main barrier to long-term successof organ transplantation. The induction of tolerance to the allografthas become a major objective and certain tolerance strategies arebeginning to be applied clinically (1). Different populations of Tregshave been described as being capable of inducing tolerance to allogeneicorgans. Most of these Tregs are CD4⁺ Tregs, while CD8⁺ Tregs are lesswell defined (2).

It has been previously described that costimulation blockade ofCD40-CD40L interaction, one of the most efficient strategies to prolongorgan allograft survival (3), induces CD8⁺CD45RC^(low) Tregs cells(called CD8⁺CD40Ig Tregs) with potent suppressive capacity (2, 4-6). Ithas been showed that donor-specific CD8⁺CD40Ig Tregs but not naturalCD8⁺CD45RC^(low) Tregs transferred tolerance to naive transplantrecipients. In addition, these cells acted in an unusual way asallograft survival was dependent on their secretion of interferon-γ(IFNγ) to enhance indoleamine 2,3-dioxygenase (IDO) expression bydendritic cells (DC) and graft endothelial cells (EC) (5). It has alsobeen recently showed that the suppressive activity of the CD8⁺CD40IgTregs was mainly performed in the presence of plasmacytoid DCs (pDCs)and that fibrinogen-like protein 2 (FGL2) was involved in thesuppression (6).

The requirement for a TCR interaction in the shaping of the regulatory Tcell population is an active and ongoing debate (2, 7). Some studiessuggest that TCR specificity and diversity is critical for in vivofunction and potency of CD8⁺ Tregs (2, 7-13). Different models for CD4⁺Tregs have shown that antigen-specific Tregs are more potent suppressorthan unrestricted Treg cells (2, 14). It is also known for CD4⁺ Tregsthat TCR diversity is critical for thymic selection and differentiationand its impact on Treg generation and function has been recentlydescribed (15). High-throughput sequencing has shown that naive Tregswith high TCR diversity expand more efficiently, are more adaptable andmore efficient in suppressing Graft versus Host Disease (GVHD) uponadoptive transfer than TCR restricted Tregs (13, 16). UsingImmunoscope®, it has been previously demonstrated that CD8⁺CD40Ig Tregsaccumulated a biased repertoire toward the Vβ11 element (5), suggestingthe possibility of a clonal expansion. To date, little is known on therecognition features of this Treg population, or of CD8⁺ Tregpopulations in general. The exact role of TCR/MHC/peptide interaction inTreg activity thus remains a topic of debate.

Moreover, there is still a great need for providing efficienttherapeutic strategies based on Treg-mediated suppression of immuneresponse against the transplant (such as donor-specific antibodies whichare associated with antibody-mediated rejection) thus avoiding the needto use non specific immunosuppressive drugs which have the drawbacks toincrease the attack rate of opportunistic diseases (e.g. bacterial,viral or fungal infections) and increase the mortality of transplantedpatients.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an isolated peptide oflength ranging between 15 and 40 amino acids which is derived from a MHCclass II molecule that comprises the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variantthereof.

In a second aspect, the invention relates to a MHC/peptide multimercomprising a peptide of length ranging between 15 and 25 amino acidsthat comprises or consists of the amino acid sequence: REEYARFDSDVGEYR(SEQ ID NO: 1) or a function-conservative variant thereof.

In a third aspect, the invention relates to an in vitro or ex vivomethod for generating a population of CD8⁺CD45RC^(low) Tregs, comprisinga step of culturing a population of CD8⁺ Tregs with a culture mediumcomprising an isolated peptide according to the invention in thepresence of a population of plasmacytoid dendritic cells.

In a fourth aspect, the invention relates to an in vitro or ex vivomethod for generating a population of CD8⁺CD45RC^(low) Tregs, comprisinga step of culturing a population of CD8+ Tregs with a culture mediumcomprising a MHC/peptide multimer according to the invention.

In a fifth aspect, the invention relates to an isolated peptide of theinvention or a MHC/peptide multimer for use as drug, for use in inducingtolerance in a patient in need thereof, and for use in preventing orreducing transplant rejection in a patient in need thereof.

In a sixth aspect, the invention relates to a pharmaceutical compositioncomprising:

a) an isolated peptide of the invention; or

b) an acid nucleic encoding a peptide of the invention; or

c) a vector comprising such nucleic acid; or

d) a host cell comprising such expression vector; or

e) a MHC/peptide multimer of the invention, or

f) an antigen-presenting cell comprising a complex MHC molecule and apeptide of the invention; or

g) a T lymphocyte that recognizes specifically the peptide of theinvention;

and a pharmaceutically acceptable carrier.

In a seventh aspect, the invention relates to an in vitro method fordetermining whether a transplanted patient (recipient) is tolerant,comprising a step of determining the presence of CD8+CD45RClow Tregs ina biological sample obtained from said transplanted patient, wherein thepresence of CD8+CD45RClow Tregs is indicative of tolerance.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have investigated whether CD8⁺CD40Ig Tregs' TCR finespecificity influences Treg function and allograft survival and havethus demonstrated for the first time in transplantation that inducedCD8⁺CD40Ig Tregs recognized a dominant peptide (called Du51) derivedfrom a polymorphic region of donor MHC class II molecules. This peptideexpanded CD8⁺ Tregs in the presence of pDCs, at least ex vivo, andinduced tolerance in naive transplanted recipients without additionaltreatment.

In addition, the inventors generated a specific tetramer anddemonstrated ex vivo and in vivo the dominant tolerance exerted byantigen-specific CD8⁺CD40Ig Tregs. Finally, they demonstrated that thispeptide was recognized by Tregs expressing Vβ11 and Vβ18-specific TCRs.These TCRs comprised a private but restricted Vβ11 repertoire in thespleen and graft, but a private and diverse Vβ18 repertoire in thespleen and a more restricted Vβ18 repertoire in the graft, that ensureefficient suppression of alloreactive immune responses.

Peptides of the Invention

A first aspect of the invention relates to an isolated peptide of lengthranging between 15 and 40 amino acids which is derived from a MHC classII molecule that comprises the amino acid sequence: REEYARFDSDVGEYR (SEQID NO: 1) (also called herein 51-18) or a function-conservative variantthereof.

In one embodiment of the invention, the peptide is an isolated peptideof length ranging between 15 and 40 amino acids which is derived from aMHC class II molecule that comprises the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variantthereof with the exclusion of the following peptides:

(SEQ ID NO: 2) NREEYARFDSDVGEYR; (SEQ ID NO: 29) QEEYVRFDSDVGEYR;(SEQ ID NO: 30) NREEFVRFDSDVGEFR; (SEQ ID NO: 31) REEFVRFDSDVGEFR;(SEQ ID NO: 32) HQEEYVRFDSDVGEYR; (SEQ ID NO: 33) HQEEYVRFDSDVGEYRA; and(SEQ ID NO: 34) HQEEYVRFDSDVGEYRAV.

As used herein, the term “function-conservative variant” refers to thosecomprise at least 70% identity with a peptide of reference (such as SEQID NO: 1), even more preferably at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5% or 99.9%, and is still able to induce tolerance andthus prevent organ rejection in substantially the same way as a peptideof reference (such as SEQ ID NO: 1 (51-18)).

In one embodiment of the invention, said peptide is derived from apolymorphic region of a donor MHC class II molecule.

In a particular embodiment, said peptide consists of the amino acidsequence: RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, the peptide of the invention comprises at least 70%identity with the peptide of SEQ ID NO: 28, even more preferably atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%, andis still able to induce tolerance and thus prevent organ rejection insubstantially the same way as the peptide of SEQ ID NO: 28.

In one embodiment of the invention, said peptide comprises at most 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39 or 40 amino acids.

In a particular embodiment of the invention, the peptide is an isolatedpeptide of 15 or 16 amino acids long that comprises or consists of theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein51-18) or a function-conservative variant thereof.

In a particular embodiment of the invention, the peptide is an isolatedpeptide of 15 or 16 amino acids long that comprises or consists of theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein51-18) or a function-conservative variant thereof with the proviso thatsaid variant is not a peptide consisting of the amino acid sequence:NREEYARFDSDVGEYR (SEQ ID NO: 2) (also called herein Du51).

As used herein, the term “peptide” refers to a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically, having no specific length. The term peptide does notexclude post-translational modifications that include but are notlimited to phosphorylation, acetylation, glycosylation and the like. Theterm also applies to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

By an “isolated” peptide, it is intended that the peptide is essentiallyfree from contaminating cellular components, such as carbohydrate,lipid, or other proteinaceous impurities associated with the polypeptidein nature. Typically, a preparation of isolated peptide contains thepeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpeptide is by the appearance of a single band followingSDS-polyacrylamide gel electrophoresis of the protein preparation andCoomassie Brilliant Blue staining of the gel. Alternatively, otheranalytical chemistry techniques such as high performance liquidchromatography (HPLC) or mass spectrometry (MS) may also be used todetermine purity. A peptide that is the predominant specie present in apreparation is substantially purified.

The invention also encompasses peptides that are function-conservativevariants of the peptide comprising SEQ ID NO: 1 as described here above.

In one embodiment, the peptide of the invention comprises at least 70%identity with the peptide of SEQ ID NO: 1, even more preferably at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%, and isstill able to induce tolerance and thus prevent organ rejection insubstantially the same way as the peptide of SEQ ID NO: 1.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the invention, it isintended that the amino acid sequence of the subject peptide isidentical to the query sequence except that the subject peptide sequencemay include up to five amino acid alterations per each 100 amino acidsof the query amino acid sequence. In other words, to obtain a peptidehaving an amino acid sequence at least 95% identical to a query aminoacid sequence, up to 5% (5 of 100) of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid.

In the frame of the application, the percentage of identity iscalculated using a global alignment (i.e., the two sequences arecompared over their entire length). Methods for comparing the identityand homology of two or more sequences are well known in the art. The“needle” program, which uses the Needleman-Wunsch global alignmentalgorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to findthe optimum alignment (including gaps) of two sequences when consideringtheir entire length, may for example be used. The needle program is forexample available on the ebi.ac.uk world wide web site. The percentageof identity in accordance with the invention is preferably calculatedusing the EMBOSS::needle (global) program with a “Gap Open” parameterequal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62matrix.

In one embodiment, the peptide according to the invention may differfrom 1, 2, 3 or 4 amino acids to the SEQ ID NO: 1 or SEQ ID NO: 2(Du51).

Such a peptide may for example only comprise substitutions compared tothe reference sequence. The substitutions preferably correspond toconservative substitutions as indicated in the table below:

Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Met,Pro, Amino acids with aliphatic hydrophobic Phe, Trp side chains Ser,Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp,Glu Amino acids with acidic side chains Lys, Arg, His Amino acids withbasic side chains Gly Neutral side chain

Typically, the invention encompasses peptides substantially identical tothe peptide consisting of SEQ ID NO: 1 (51-18) or SEQ ID NO: 2 (Du51) inwhich one or more residues have been conservatively substituted with afunctionally similar residue and which displays the functional aspectsof the peptides consisting of SEQ ID NO: 1 (51-18) or SEQ ID NO: 2(Du51) as described here below, i.e. being still able to inducetolerance and thus prevent organ rejection in substantially the same wayas a peptide consisting of the given amino acid sequence (e.g. thepeptides 51-18 or Du51).

The term “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a peptide having one or more residueschemically derivatized by reaction of a functional side group. Examplesof such derivatized molecules include for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Chemical derivatives also include peptides that contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For examples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. The term “conservativesubstitution” also includes the use of non natural amino acids aimed tocontrol and stabilize peptides or proteins secondary structures. Thesenon natural amino acids are chemically modified amino acids such asprolinoamino acids, beta-amino acids, N-methylamino acids,cyclopropylamino acids, alpha,alpha-substituted amino acids as describehere below. These non natural amino acids may include also fluorinated,chlorinated, brominated- or iodinated modified amino acids.

To verify whether the newly generated peptides induce the samebiological properties than the initially characterized peptides 51-18 orDu51, an analysis of CD8⁺CD45RC^(low) Treg activation and/or an analysisof tolerance induction after in vivo peptide infusion (such as describedin the section EXAMPLE) may be performed with each peptide.Additionally, a time-course and a dose-response performed in a model ofallograft will determine the optimal conditions for each peptide.

In one embodiment, the peptide consists of the amino acid sequenceselected from the group consisting of:

(SEQ ID NO: 3) REEYARFDSDVGEFR, (SEQ ID NO: 4) REEYVRFDSDVGEYR,(SEQ ID NO: 5) QEEYARFDSDVGEYR, or (SEQ ID NO: 6) REEYARFDSDVGVYR.

In one embodiment, the peptide having the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) bears an asparagine (N) at theN-terminus.

In a preferred embodiment, the peptide consists of the amino acidsequence selected from the group consisting of:

(SEQ ID NO: 7) NREEYARFDSDVGEFR, (SEQ ID NO: 8) NREEYVRFDSDVGEYR,(SEQ ID NO: 9) NQEEYARFDSDVGEYR, or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

According to the invention, the peptides of the invention may beproduced by conventional automated peptide synthesis methods or byrecombinant expression. General principles for designing and makingproteins are well known to those of skill in the art.

Peptides of the invention may be synthesized in solution or on a solidsupport in accordance with conventional techniques. Various automaticsynthesizers are commercially available and can be used in accordancewith known protocols as described in Stewart and Young; Tam et al.,1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer,1979. Peptides of the invention may also be synthesized by solid-phasetechnology employing an exemplary peptide synthesizer such as a Model433A from Applied Biosystems Inc. The purity of any given peptide;generated through automated peptide synthesis or through recombinantmethods may be determined using reverse phase HPLC analysis. Chemicalauthenticity of each peptide may be established by any method well knownto those of skill in the art. As an alternative to automated peptidesynthesis, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of choice is inserted intoan expression vector, transformed or transfected into an appropriatehost cell and cultivated under conditions suitable for expression asdescribed herein below. Recombinant methods are especially preferred forproducing longer peptides (polypeptides).

Nucleic Acids, Vectors and Recombinant Host Cells

Alternatively, a nucleic acid encoding a peptide of the invention (suchas the peptide shown in SEQ ID NO: 1), a vector comprising such nucleicacid or a host cell comprising such expression vector are also ofinterest within the context of the invention.

In one embodiment, the peptide consists of an amino acid sequencedescribed above.

Nucleic acids of the invention may be produced by any technique knownper se in the art, such as, without limitation, any chemical,biological, genetic or enzymatic technique, either alone or incombination(s).

In its broadest sense, a “vector” is any vehicle capable of facilitatingthe transfer of a nucleic acid to the cells In general, the vectorsuseful in the invention include, but are not limited to, plasmids,phagemids, viruses, other vehicles derived from viral or bacterialsources that have been manipulated by the insertion or incorporation ofthe nucleic acid sequences of interest. Viral vectors are a preferredtype of vector and include, but are not limited to nucleic acidsequences from the following viruses: retrovirus, such as moloney murineleukemia virus, harvey murine sarcoma virus, murine mammary tumor virus,and mouse sarcoma virus; adenovirus, adeno-associated virus; SV40-typeviruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses;herpes virus; vaccinia virus; polio virus; and RNA virus such as aretrovirus. One can readily employ other vectors not named but known tothe art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired peptides, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in KRIEGLER (ALaboratory Manual,” W.H. Freeman C.O., New York, 1990) and in MURRY(“Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Cliffton,N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses andadeno-associated viruses, which are double-stranded DNA viruses thathave already been approved for human use in gene therapy. Theadeno-associated virus can be engineered to be replication deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g., SANBROOK et al., “Molecular Cloning: A LaboratoryManual,” Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, 5V40, andpBlueScript. Other plasmids are well known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids may be delivered by a variety of parenteral, mucosal andtopical routes. For example, the DNA plasmid can be injected byintramuscular, intradermal, subcutaneous, or other routes. It may alsobe administered by intranasal sprays or drops, rectal suppository andorally. It may also be administered into the epidermis or a mucosalsurface using a gene-gun. The plasmids may be given in an aqueoussolution, dried onto gold particles or in association with another DNAdelivery system including but not limited to liposomes, dendrimers, andmicroencapsulation.

According to the invention, examples of host cells that may be used areantigen-presenting cells (APC) such as human dendritic cells ormonocytes (particularly those obtained from the patient to be treated).

The means by which the vector carrying the gene may be introduced intothe cells include, but are not limited to, microinjection,electroporation, transduction, or transfection using DEAE-dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art.

MHC/Peptide Multimers

Another aspect of the invention is a MHC/peptide multimer comprising apeptide of length ranging between 15 and 25 amino acids that comprisesor consists of the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1)(also called herein 51-18) or a function-conservative variant thereof asdescribed here above.

In one embodiment of the invention, said peptide comprises at most 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

In a particular embodiment, said MHC/peptide multimer comprises apeptide of 15 or 16 amino acids long that comprises or consists of theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein51-18) or a function-conservative variant thereof as described hereabove.

As used herein, the term “MHC/peptide multimer” refers to a stablemultimeric complex composed of major histocompatibility complex (MHC)protein subunits loaded with a peptide of the invention. According tothe invention, said MHC/peptide multimer (also called herein MHC/peptidecomplex) include, but are not limited to, a MHC/peptide dimer, trimer,tetramer or pentamer.

The term “Major Histocompatibility Complex” (MHC) is a genericdesignation meant to encompass the histo-compatibility antigen systemsdescribed in different species including the human leucocyte antigens(HLA).

In one embodiment of the invention, the MHC/peptide multimer is a MHCclass I/peptide multimer.

In a particular embodiment of the invention, said MHC/peptide multimeris a rat MHC RT1.A^(a)/peptide multimer. It should be further noted thatnonclassical rat MHC class I molecules are also encompassed within thecontext of the invention.

In another particular embodiment, the MHC/peptide multimer is a HLAcorresponding to MHC class I/peptide multimer.

In humans there are three major different genetic loci that encode MHCclass I molecules (the MHC-molecules of the human are also designatedhuman leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01,HLA-A*02, and HLA-A*11 are examples of different MHC class I allelesthat can be expressed from these loci.

It should be further noted that nonclassical human MHC class I moleculessuch as HLA-E (functional homolog in mice is called Qa-1b) and MICA/Bmolecules are also encompassed within the context of the invention.

Accordingly, the MHC/peptide multimer is a HLA/peptide multimer selectedfrom the group consisting of HLA-A/peptide multimer, HLA-B/peptidemultimer, HLA-C/peptide multimer, HLA-E/peptide multimer, MICA/peptidemultimer and MICB/peptide multimer.

In one embodiment, said isolated peptide is selected from the groupconsisting of:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8) NREEYVRFDSDVGEYR;(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

Methods for obtaining MHC/peptide tetramers are described in WO96/26962and WO01/18053, which are incorporated by reference.

In one embodiment of the invention, said MHC/peptide multimer can beused to visualise T cell populations that are specific for the MHC classI RT1.A^(a)/peptide complex or a HLAs corresponding to MHC classI/peptide complex as described here above.

The MHC/peptide multimer may be a multimer where the heavy chain of theMHC is biotinylated, which allows combination as a tetramer withstreptavidine. Such MHC-peptide tetramer has an increased avidity forthe appropriate TCR-carrier T lymphocytes and can therefore be used tovisualize reactive populations by immunofluorescence.

In another embodiment of the invention, said MHC/peptide multimer can beused for the detection and/or isolation by screening (in flow cytometryor by immunomagnetic screening) of T cell populations that are specificfor a MHC/peptide complex as described here above.

In a particular embodiment of the invention, a MHC class IRT1.A^(a)/peptide multimer or a HLA corresponding to MHC class I/peptidemultimer can be used for the detection and/or isolation by screening (inflow cytometry or by immunomagnetic screening) of T cell populationsthat are specific for said MHC class I RT1.A^(a)/peptide complex or saidHLA corresponding to MHC class I/peptide multimer as described hereabove.

Another aspect of the invention is beads, microspheres or nanoparticlescoated with a MHC/peptide multimer as described here above.

Antigen Presenting Cells and Uses Thereof

Another aspect of the invention is a peptide-presenting cell comprisinga MHC/peptide multimer of the invention as described here above.

In one embodiment of the invention, said multimer MHC/peptide is a MHCclass I RT1.A^(a)/peptide multimer.

In another particular embodiment, the MHC/peptide multimer is a HLAcorresponding to MHC class I/peptide multimer as defined above.

Accordingly, the MHC/peptide multimer is a HLA/peptide multimer selectedfrom the group consisting of HLA-A/peptide multimer, HLA-B/peptidemultimer, HLA-C/peptide multimer, HLA-E/peptide multimer, MICA/peptidemultimer and MICB/peptide multimer.

In one embodiment, said peptide is selected from the group consistingof:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8) NREEYVRFDSDVGEYR;(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

In one embodiment of the invention, said antigen presenting cell isderived from the patient to be treated.

As used herein, the terms “antigen-presenting cell” (APC) also calledherein “peptide-presenting cell” are used interchangeably and refer to aclass of immune cells capable of internalizing and processing anantigen, so that antigenic determinants are presented on the surface ofthe cell as MHC-associated complexes, in a manner capable of beingrecognized by the immune system (e. g., MHC class I restricted cytotoxicT lymphocytes and/or MHC class II restricted helper T lymphocytes). Thetwo requisite properties that allow a cell to function as an APC are theability to process endocytosed antigens and the expression of MHC geneproducts. Examples of APC include dendritic cells (DC), mononuclearphagocytes (e. g. macrophages), B lymphocytes, Langerhans cells of theskin and, in humans, endothelial cells.

In one embodiment of the invention, said APC is a plasmacytoid dendriticcell (pDC).

As used herein, the term “plasmacytoid dendritic cells” (pDC) refers toinnate immune cells that circulate in the blood and are found inperipheral lymphoid organs. They constitute a group of cells belongingto the peripheral blood mononuclear cells (PBMC) group. Human pDCstypically express the surface markers IL-3 receptor a chain (IL-3Ra,CD123), BDCA-2 (CD303) and BDCA-4 (CD304), but do not express CD11c orCD14, which distinguishes them from conventional dendritic cells ormonocytes.

In one embodiment of the invention, said pDC is a mature pDC. MaturepDCs typically express the surface markers HLA-DR, CD86 and producelarge amount of IFNalpha and IFNbeta.

In order to prepare such APCs of the invention, cells having anantigen-presenting ability are isolated from the patient to be treated,and pulsed ex vivo with a peptide of the invention to form a complexwith the MHC molecule.

In case dendritic cells are used, the APC of the invention can beprepared as follows. Lymphocytes are isolated from peripheral blood ofthe patient to be treated by Ficoll method; adherent cells are separatedfrom non-adherent cells; the adherent cells are then cultured in thepresence of GM-CSF and IL-4 to induce dendritic cells; and the dendriticcells are pulsed by culturing with a peptide of the invention to obtainthe APCs of the invention. The dendritic cells should be exposed to thepeptide for sufficient time to allow the antigen to be internalized andpresented on the dendritic cells surface. The resulting dendritic cellscan then be re-administrated to the patient to be treated. Such methodsare described in WO93/208185 and EP0563485, which are incorporated byreference.

Lymphocytes T and Uses Thereof

Another aspect of the invention is a T lymphocyte that recognizesspecifically the peptide of 15 or 16 amino acids long that comprises orconsists of the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or afunction-conservative variant thereof of the invention.

In one embodiment, said peptide is selected from the group consistingof:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8) NREEYVRFDSDVGEYR;(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

In one embodiment of the invention, said T lymphocyte is a regulatory Tlymphocyte (Treg). In another embodiment of the invention, said Tlymphocyte is a T reg clone.

As used herein, the terms “regulatory T cell” or “regulatory Tlymphocyte” are used interchangeably and refer to a specific populationof T lymphocytes that have the capacity to dominantly suppress theproliferation of responder T cells in vitro and inhibit diseases andconditions associated with or caused by an excessive immune responsesuch as for example autoimmune diseases, transplantation rejection or agraft versus host disease. Treg were originally identified as aCD4⁺CD25⁺ cell population, but are also characterized by the expressionof the forkhead family transcription factor, FoxP3. More recently, CD8⁺Treg cells have also been identified as previously described.

In a particular embodiment, said Treg is a CD8⁺CD45RC^(low) Treg.

In another embodiment, said T lymphocyte is a genetically modified Tlymphocyte that expresses a TCR that recognizes specifically the peptideof the invention.

Accordingly, the T lymphocyte expresses a biased restricted TCRrepertoire. Biased (TCR) is well known form the skilled man in the art.Examples of TCR bias have been observed in classical polymorphic majorhistocompatibility complex (MHC)-restricted immune responses and havebeen described (64).

Methods for Obtaining a Population of Regulatory T Cells

In another aspect, the invention relates to an in vitro or ex vivomethod for generating a population of Tregs, comprising a step ofculturing a population of Tregs with a culture medium comprising anisolated peptide of length ranging between 15 and 40 amino acids whichis derived from a MHC class II molecule that comprises the amino acidsequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservativevariant thereof according the invention in the presence of a populationof dendritic cells.

In one embodiment, said peptide consists of the amino acid sequence:RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, the invention relates to an in vitro or ex vivomethod for generating a population of CD8⁺CD45RC^(low) Tregs, comprisinga step of culturing a population of CD8⁺ Tregs with a culture mediumcomprising an isolated peptide of the invention in the presence of apopulation of plasmacytoid dendritic cells (pDCs).

In one embodiment, pDCs are mature pDCs.

In one embodiment, said isolated peptide is an isolated peptide of 15 or16 amino acids long that comprises or consists of the amino acidsequence: REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein 51-18) or afunction-conservative variant thereof

In a particular embodiment, said isolated peptide selected from thegroup consisting of:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8) NREEYVRFDSDVGEYR;(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

As used herein, the term “culture medium” refers to any medium capableof supporting the growth and the differentiation of T cells intoregulatory T cells. Typically, it consists of a base medium containingnutrients (a source of carbon, aminoacids), a pH buffer and salts, whichcan be supplemented with growth factors and/or antibiotics. Typically,the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium,all of which are commercially available standard media.

Preferred media formulations that will support the growth and thedifferentiation of naive T cells into regulatory T cells includechemically defined medium (CDM). As used herein, the term “chemicallydefined medium” (CDM) refers to a nutritive solution for culturing cellswhich contains only specified components, preferably components of knownchemical structure. A chemically defined medium is a serum-free andfeeder-free medium.

The step of culturing a population of CD8⁺ Tregs with the peptide of theinvention in the presence of a population of pDCs shall be carried outfor the necessary time required for the presentation of said peptide bythe pDCs to the CD8⁺ Tregs.

Typically, the culture of a population of CD8⁺ Tregs with the peptide ofthe invention in the presence of a population of pDCs shall be carriedfrom 1 day to 1 week or more.

In a particular embodiment, the method may comprise an additional stepof isolating the population of regulatory T cells thus generated.

Alternatively, the invention relates to an in vitro or ex vivo methodfor generating a population of Tregs, comprising a step of culturing apopulation of Tregs with a culture medium comprising a MHC/peptidemultimer of the invention as defined above.

In one embodiment, the invention relates to an in vitro or ex vivomethod for generating a population of CD8⁺CD45RC^(low) Tregs, comprisinga step of culturing a population of CD8⁺ Tregs with a culture mediumcomprising a MHC/peptide multimer of the invention as defined above.

In one embodiment, the multimer is coated on a nanoparticle. Thus, thenanoparticle displays at its surface the MHC/peptide multimer accordingto the invention.

Within the context of the invention, the nanoparticles are of smallsize, small enough to be recognized by Tregs. In preferred embodiments,the nanoparticles have a core with a mean diameter between 0.5 and 10nm, more preferably between 1 and 2.5 nm.

The core of the nanoparticle may be a polymeric core. Preferably, thenanoparticle comprises polymers are selected from the group consistingof carbohydrate-based polymers (e.g., cellulose-based nanoparticles,chitosan-based nanoparticles), polyethylene glycol (PEG), polypropyleneglycol (PPG), and copolymers of PEG and PPG, branched copolymerscontaining PEG and caprolactone, PEG and lactide, and PEG and[lactide-co-glycolide].

The core of the nanoparticle may also be a metallic core. Preferably,the metallic core comprises Au, Ag or Cu, for example an alloy selectedfrom Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd, Au/Ag/Cu/Pd, Au/Fe, Au/Cu,Au/Gd, Au/Fe/Cu, Au/Fe/Gd or Au/Fe/Cu/Gd.

Preferably, the nanoparticles are soluble in most organic solvents andespecially water. Nanoparticles can be prepared according to techniqueswell-known in the art.

In one embodiment, the invention relates to an in vitro or ex vivomethod for generating a population of CD8⁺CD45RC^(low) Tregs, comprisinga step of culturing a population of CD8⁺ Tregs with a culture mediumcomprising nanoparticles coated with a MHC/peptide multimer of theinvention as defined above.

In another aspect, the invention relates to a population of Tregs, moreparticularly a population of CD8⁺CD45RC^(low) Tregs, obtained by any oneof the methods as previously described.

Pharmaceutical Compositions

Another aspect of the invention relates to a pharmaceutical compositioncomprising:

-   -   a) an isolated peptide of length ranging between 15 and 40 amino        acids which is derived from a MHC class II molecule that        comprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID        NO: 1) or a function-conservative variant thereof; or    -   b) an acid nucleic encoding a peptide of the invention; or    -   c) a vector comprising such nucleic acid; or    -   d) a host cell comprising such expression vector; or    -   e) a complex MHC molecule and a peptide of the invention; or    -   f) an antigen-presenting cell comprising a complex MHC molecule        and a peptide of the invention; or    -   g) a T lymphocyte that recognizes specifically the peptide of        the invention;    -   and a pharmaceutically acceptable carrier.

In a particular embodiment, said peptide consists of the amino acidsequence: RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, said pharmaceutical composition comprises an isolatedpeptide of 15 or 16 amino acids long that comprises or consists of theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or afunction-conservative variant thereof

In a particular embodiment, said pharmaceutical composition comprises anisolated peptide selected from the group consisting of:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8) NREEYVRFDSDVGEYR;(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

Any therapeutic agent of the invention as above described may becombined with pharmaceutically acceptable excipients, and optionallysustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, intranasal, intraocular, intravenous, intramuscular orsubcutaneous administration. Preferably, the pharmaceutical compositionscontain vehicles which are pharmaceutically acceptable for a formulationcapable of being injected. These may be in particular isotonic, sterile,saline solutions (monosodium or disodium phosphate, sodium, potassium,calcium or magnesium chloride and the like or mixtures of such salts),or dry, especially freeze-dried compositions which upon addition,depending on the case, of sterilized water or physiological saline,permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment. For example, it is well within the skillof the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the subject to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 7 mg/kg of body weight per day.

To prepare pharmaceutical compositions, an effective amount of apolypeptide or a nucleic acid according to the invention may bedissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The peptides according to the invention and other therapeutic agent ofthe invention can be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution may be suitably buffered and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. In this connection,sterile aqueous media which can be employed will be known to those ofskill in the art in light of the present disclosure. For example, onedosage could be dissolved in 1 ml of isotonic NaCl solution and eitheradded to 1000 ml of hypodermoclysis fluid or injected at the proposedsite of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; time release capsules; and any other form currentlyused.

Therapeutic Methods and Uses

The invention provides methods and compositions (such as pharmaceuticalcompositions) for use in inducing immune tolerance in a patient in needthereof.

The invention also provides methods and compositions for use inpreventing or reducing transplant rejection in a patient in needthereof.

Accordingly, the invention relates to an isolated peptide of lengthranging between 15 and 40 amino acids which is derived from a MHC classII molecule that comprises the amino acid sequence: REEYARFDSDVGEYR (SEQID NO: 1) (also called herein 51-18) or a function-conservative variantthereof as defined above for use as drug.

In one embodiment, said peptide consists of the amino acid sequence:RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, said peptide is an isolated peptide of 15 or 16 aminoacids long that comprises or consists of the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant.

In another aspect, the invention relates to an isolated peptide oflength ranging between 15 and 40 amino acids which is derived from a MHCclass II molecule that comprises the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein 51-18) or afunction-conservative variant thereof as defined above for use ininducing immune tolerance in a patient in need thereof.

In one embodiment, said peptide consists of the amino acid sequence:RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, said peptide is an isolated peptide of 15 or 16 aminoacids long that comprises or consists of the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant foruse in inducing immune tolerance in a patient in need thereof.

As used herein, the term “immune tolerance” refers to a state ofunresponsiveness of the immune system to substances or tissues that havethe capacity to elicit an immune response. Peptides of the invention areuseful for achieving tolerance or partial tolerance against thetransplant upon transplantation of said transplant. As used herein, a“partial tolerance” is a partial immune tolerance results in a reducedimmune response.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, in addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages. Immune cells involved in the immuneresponse include lymphocytes, such as B cells and T cells (CD4⁺, CD8⁺,Th1 and Th2 cells); antigen presenting cells (e.g. professional antigenpresenting cells such as dendritic cells); natural killer cells; myeloidcells, such as macrophages, eosinophils, mast cells, basophils, andgranulocytes.

For instance, immune responses are involved in transplant rejection, aswell as in the concomitant physiological result of such immuneresponses, such as for example, interstitial fibrosis, chronic graftarteriosclerosis, or vasculitis.

Thus, treated patients with a peptide of the invention (as well as witha MCH/peptide mutimer of the invention, or peptide-specific Tregs asdefined herein) in comparison with untreated patients, display thefollowing physiological features: a) a decreased level of an immuneresponse against the transplant (thought to be mediated at least in partby B cell mediated immune responses, more particularly donor-specificantibodies); b) a delay in the onset or progression of a immune responseagainst the transplant; or c) a reduced risk of the onset or progressionof an immune response against the transplant.

By “patient in need thereof” is meant an individual suffering from orsusceptible of suffering from transplant rejection to be treated. Theindividuals to be treated in the frame of the invention are mammals,preferably human beings.

In still another aspect, the invention relates to an isolated peptide oflength ranging between 15 and 40 amino acids which is derived from a MHCclass II molecule that comprises the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) (also called herein 51-18) or afunction-conservative variant thereof as defined above for use inpreventing or reducing transplant rejection in a patient in needthereof.

In one embodiment, said peptide consists of the amino acid sequence:RLLARLIYNREEYARFDSDVGEYRAVTELGRPSAEYRNKQ (SEQ ID NO: 28) or afunction-conservative variant thereof.

In one embodiment, said peptide is an isolated peptide of 15 or 16 aminoacids long that comprises or consists of the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1) or a function-conservative variant.

As used herein, the term “preventing or reducing transplant rejection”is meant to encompass prevention or inhibition of immune transplantrejection, as well as delaying the onset or the progression of immunetransplant rejection. The term is also meant to encompass prolongingsurvival of a transplant in a patient, or reversing failure of atransplant in a patient. Further, the term is meant to encompassameliorating a symptom of an immune transplant rejection, including, forexample, ameliorating an immunological complication associated withimmune rejection, such as for example, interstitial fibrosis, chronicgraft arteriosclerosis, or vasculitis.

As used herein, the term “transplant rejection” encompasses both acuteand chronic transplant rejection. “Acute rejection” is the rejection bythe immune system of a tissue transplant recipient when the transplantedtissue is immunologically foreign. Acute rejection is characterized byinfiltration of the transplant tissue by immune cells of the recipient,which carry out their effector function and destroy the transplanttissue. The onset of acute rejection is rapid and generally occurs inhumans within a few weeks after transplant surgery. Generally, acuterejection can be inhibited or suppressed with immunosuppressive drugssuch as rapamycin, cyclosporin and the like. “Chronic rejection”generally occurs in humans within several months to years afterengraftment, even in the presence of successful immunosuppression ofacute rejection. Fibrosis is a common factor in chronic rejection of alltypes of organ transplants.

The term “transplantation” and variations thereof refers to theinsertion of a transplant (also called graft) into a recipient, whetherthe transplantation is syngeneic (where the donor and recipient aregenetically identical), allogeneic (where the donor and recipient are ofdifferent genetic origins but of the same species), or xenogeneic (wherethe donor and recipient are from different species). Thus, in a typicalscenario, the host is human and the graft is an isograft, derived from ahuman of the same or different genetic origins. In another scenario, thegraft is derived from a species different from that into which it istransplanted, including animals from phylogenically widely separatedspecies, for example, a baboon heart being transplanted into a humanhost.

In one embodiment the donor of the transplant is a human. The donor ofthe transplant can be a living donor or a deceased donor, namely acadaveric donor.

In one embodiment, the transplant is an organ, a tissue or cells.

As used herein, the term “organ” refers to a solid vascularized organthat performs a specific function or group of functions within anorganism. The term organ includes, but is not limited to, heart, lung,kidney, liver, pancreas, skin, uterus, bone, cartilage, small or largebowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube,gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limbincluding upper and lower, spleen, stomach, testes, thymus, thyroid,trachea, ureter, urethra, uterus. As used herein, the term “tissue”refers to any type of tissue in human or animals, and includes, but isnot limited to, vascular tissue, skin tissue, hepatic tissue, pancreatictissue, neural tissue, urogenital tissue, gastrointestinal tissue,skeletal tissue including bone and cartilage, adipose tissue, connectivetissue including tendons and ligaments, amniotic tissue, chorionictissue, dura, pericardia, muscle tissue, glandular tissue, facialtissue, ophthalmic tissue.

In a particular embodiment of the invention, the transplant is a cardiacallotransplant.

As used herein, the term “cells” refers to a composition enriched forcells of interest, preferably a composition comprising at least 30%,preferably at least 50%, even more preferably at least 65% of saidcells.

In certain embodiments the cells are selected from the group consistingof multipotent hematopoietic stem cells derived from bone marrow,peripheral blood, or umbilical cord blood; or pluripotent (i.e.embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) ormultipotent stem cell-derived differentiated cells of different celllineages such as cardiomyocytes, beta-pancreatic cells, hepatocytes,neurons, etc. . . . .

In one embodiment, the cell composition is used for allogeneichematopoietic stem cell transplantation (HSCT) and thus comprisesmultipotent hematopoietic stem cells, usually derived from bone marrow,peripheral blood, or umbilical cord blood.

HSCT can be curative for patients with leukemia and lymphomas. However,an important limitation of allogeneic HCT is the development of graftversus host disease (GVHD), which occurs in a severe form in about30-50% of humans who receive this therapy.

Peptides of the invention are useful in inducing immune tolerance andthus in preventing or reducing Graft-versus-Host-Disease (GvHD).

Accordingly, in one embodiment, the patient in need thereof is affectedwith a disease selected from the group consisting of acute myeloidleukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia(CML); myelodysplasia syndrome (MDS)/myeloproliferative syndrome;lymphomas such as Hodgkin and non-Hodgkin lymphomas, chronic lymphaticleukemia (CLL) and multiple myeloma.

In another aspect, the invention relates to

a) an acid nucleic encoding a peptide of the invention; or

b) a vector comprising such nucleic acid; or

c) a host cell comprising such expression vector; or

d) a MHC/peptide multimer of the invention; or

e) an antigen-presenting cell comprising a complex MHC molecule and apeptide of the invention; or

f) a T lymphocyte that recognizes specifically the peptide of theinvention; for use in inducing immune tolerance in a patient in needthereof.

In still another aspect, the invention relates to

a) an acid nucleic encoding a peptide of the invention; or

b) a vector comprising such nucleic acid; or

c) a host cell comprising such expression vector; or

d) a MHC/peptide multimer of the invention; or

e) an antigen-presenting cell comprising a complex MHC molecule and apeptide of the invention; or

f) a T lymphocyte that recognizes specifically the peptide of theinvention; for use in preventing or reducing transplant rejection in apatient in need thereof.

Another aspect of the invention relates to a method for inducing immunetolerance in a patient in need thereof, comprising a step ofadministering to said patient a prophylactically effective amount of anisolated peptide of the invention as described above, or a nucleic acidof the invention, or an expression vector of the invention, or a hostcell of the invention, or a MHC/peptide multimer of the invention, or aT lymphocyte that recognizes specifically the peptide of the invention.

Another aspect of the invention relates to a method for preventing orreducing transplant rejection in a patient in need thereof, comprising astep of administering to said patient a prophylactically effectiveamount of an isolated peptide of the invention as described above, or anucleic acid of the invention, or an expression vector of the invention,or a host cell of the invention, a MHC/peptide multimer of the inventionor a T lymphocyte that recognizes specifically the peptide of theinvention.

As used herein, the term “prophylactically effective amount” is intendedfor a minimal amount of active agent, which is necessary to prevent,reduce, alleviate or delay) transplant rejection in a patient.

Diagnostic Methods of the Invention

In a still further aspect, the invention relates to an in vitro methodfor determining whether a transplanted patient (recipient) is tolerant,comprising a step of determining the presence of CD8+CD45RC^(low) Tregsin a biological sample obtained from said transplanted patient, whereinthe presence of CD8+CD45RC^(low) Tregs is indicative of tolerance.

As used herein, the term “determining” includes qualitative and/orquantitative detection (i.e. detecting and/or measuring the amount) withor without reference to a control or a predetermined value. As usedherein, “detecting” means determining if CD8+CD45RC^(low) Tregs arepresent or not in a biological sample and “measuring” means determiningthe amount of CD8+CD45RC^(low) Tregs in a biological sample.

As used herein, the term “biological sample” has its general meaning inthe art and refers to any sample which may be obtained from a patientfor the purpose of in vitro evaluation. A preferred biological sample isa blood sample (e.g. whole blood sample, serum sample, or plasmasample).

For example, the determination of the presence of CD8+CD45RC^(low) Tregsmay comprise a step of contacting the biological sample with selectivereagents such as antibodies, and thereby detecting the presence, ormeasuring the amount, of cells of interest originally in said biologicalsample. Contacting may be performed in any suitable device, such as aplate, microtiter dish, test tube, well, glass, column, and so forth.

Accordingly, the methods according to the invention comprise a step ofcontacting the biological sample with a binding partner capable ofselectively interacting with CD8+CD45RC^(low) Tregs in said biologicalsample.

In one embodiment of the invention, the binding partner is a MHC/peptidemultimer comprising a peptide of the invention as described here above.

In a preferred embodiment, the MHC/peptide multimer is a MHC/peptidetetramer comprising a peptide selected from the group consisting of:

(SEQ ID NO: 1) REEYARFDSDVGEYR; (SEQ ID NO: 2) NREEYARFDSDVGEYR;(SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4) REEYVRFDSDVGEYR;(SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6) REEYARFDSDVGVYR;(SEQ ID NO: 7) NREEYARFDSDVGEFR, (SEQ ID NO: 8) NREEYVRFDSDVGEYR,(SEQ ID NO: 9) NQEEYARFDSDVGEYR or (SEQ ID NO: 10) NREEYARFDSDVGVYR.

In one embodiment, the patient is a mammal, preferably a human being.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Analysis of CD8⁺CD45RC^(low) Tregs activation in response todonor-derived peptide stimulation. (A) CD8⁺ Tregs were cocultured for 6days with syngeneic CpG-matured pDCs in the presence of peptides. Foreach experiment, the percentage of CD25 positive Tregs after 6 days ofcoculture with pDCs only, was given the value 1. Mean value 1 is equalto 32.85±1.98%. Results are expressed as the ratio±SEM between thepercentage of CD25 positive cells after peptide stimulation andpercentage of CD25 positive cells in the control condition withoutpeptide. *p<0.05, **p<0.01 and ***p<0.001 versus control condition(value 1.0). n=4 to 18 for each peptide. (B) Analysis of Treg activationin response to Du51 shorter peptide derivatives. On the left, 18Du51-derivatives are detailed and classified by aa sequence length, from9 aa to 15 aa. The box highlights mismatched aa between the donor andrecipient. On the right, Treg activation in response to Du51-derivativeswas analyzed by CD25 expression. CD8⁺ Tregs were cocultured for 6 dayswith syngeneic CpG-matured pDCs in the presence of each peptide. Barsrepresent the ratio between the percentage of CD25 positive cells afterpeptide stimulation and percentage of CD25 positive cells in the controlcondition without peptide. *p<0.05, **p<0.01 and ***p<0.001 versus Du51condition. n=3 to 14 for each peptide.

FIG. 2: In vitro and in vivo suppressive potential of Du51-specificCD40IgCD8⁺ Tregs. (A) The proliferation of naive CFSE-labeled LEW.1ACD4⁺CD25⁻ T cells stimulated with either donor LEW.1W (direct pathway)or alloantigens-loaded recipient LEW.1A (indirect pathway) pDCs wasanalyzed after 6 days of culture, in the absence or presence of LEW.1Anaive, total, tetramer⁻ or tetramer⁺ CD8⁺CD40Ig Tregs at a 1:1effector:suppressor ratio. The CD4⁺CD25⁻ T cell proliferation alone was80% and was given the value 100 in each experiment. Graphs represent themean±SEM of relative CD4⁺CD25⁻ T cell proliferation. *p<0.05. n=4. (B)2.5×10⁶ total or tetramer⁻ CD8⁺CD40Ig Tregs were injected i.v intosublethally irradiated recipients (LEW.1A) the day before heartallotransplantation (LEW.1W). Graft survival was assessed by abdominalpalpation of cardiac beating. p<0.01 for total (n=4) versus tetramer⁻CD8⁺CD40Ig Tregs (n=3).

FIG. 3: Tolerance induction after in vivo peptide Du51 infusion. (A)Recipients were either untreated (n=9), treated with 5 single i.vinjections of 0.5 mg peptide at day −6, −3 pre-graft, day 0, +3 and +7post-graft (n=4), or treated with continuous infusion of peptide by i.pmini osmotic pumps (ALZET), delivering from day-7 and for 28 days,either 20.83 μg/hour alone (Du51 0.5 mg/day: n=8) or combined with adepleting anti-CD8 mAb (OX8) (n=6) or an anti-MHC class I mAb (OX18)(n=5), or 40.66 μg/hour in the LEW.1W/LEW.1A (Du51 1 mg/day: n=5) orBN/LEW.1A (Du51 1 mg/day (BN/1A): n=4) strains combination. **p<0.01Du51 0.5 mg/day versus untreated animals and control peptide 0.5 mg/day.*p<0.05 Du51 0.5 mg/day+depleting anti-CD8 mAb versus Du51 0.5 mg/day.***p<0.001 Du51 1 mg/day versus untreated animals. **p<0.01 Du51 1mg/day versus Du51 1 mg/day in BN/1A combination. *p<0.05 Du51 1 mg/dayversus Du51 0.5 mg/day. (B) Alloantibody production was evaluated innaive (n=3), grafted syngeneic (n=3), grafted untreated (n=3),CD40Ig-treated (n=3), long-term Du51-treated (n=2) and rejectedDu51-treated (n=4) animals. Sera were collected <30 days after rejectionor >120 days after transplantation. Sera were incubated with donorsplenocytes and analyzed by flow cytometry for IgG, IgG1, IgG2a or IgG2bAbs production. Graph represents MFI±SEM. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. (C) Spleens were recovered after rejection or at day 120and cell populations were stained and analyzed by flow cytometry. Graphrepresents the absolute number of each sub-population in the totalspleen from either (n=12), long-term Du51-treated (n=2) or rejectedDu51-treated recipients (n=5). (D) Spleen of naive (n=4), long-termDu51-treated (n=2) or rejected Du51-treated (n=4) recipients wereincubated with RT1.A^(a)/Du51 tetramers labeled with streptavidinconjugated to PE and APC. Results are plotted into graphs for percentageof tetramer⁺ cells among CD8⁺ Tregs and absolute number of tetramer⁺CD8⁺ Tregs in spleen ±SEM.

FIG. 4: Splenocyte-mediated transfer of tolerance following Du51 peptidemonotherapy. A. LEW.1A recipients were sublethally irradiated (4.5 Gy)at day 1 and received heart allografts and i.v. injections of 1.5·10⁸splenocytes from long surviving recipient or naive animals at day 0.Graft survival was monitored by abdominal palpation. B. IgG alloantibodyproduction was evaluated in naive (n 3), untreated recipients that hadrejected their graft (n=3) and 1^(st) spl-transferred long-termrecipients more than 120 days after transplantation.

EXAMPLE MHCII⁺ Allopeptide Induce Tolerance

Material & Methods

Animals and Cardiac Transplantation Models:

Allotransplantations of heart were performed between whole MHCincompatible LEW.1W (RT1.A^(u) as donors) and LEW.1A (RT1.A^(a) asrecipients) male rats as previously described (5). The experiments wereapproved by the regional ethical committee for animal experimentation.

Adenovirus-Mediated Gene Transfer:

The Ad encoding for the extracellular portion of mouse CD40 fused to theconstant domains of human IgG1 (AdCD40Ig) and the recombinant non-codingadenovirus (Ad) Addl324, as well as the procedure of intragraftdelivery, have been described previously (5). Briefly, adenoviralparticles (2·10¹⁰ infectious particles) were slowly injected at 3 pointsinto the cardiac ventricular walls.

Peptides Libraries:

16-mer overlapping peptide libraries with 4 aa lagging were designed tocover the entire polymorphic sequences of MHC-I RT1.Au (alpha 1, 2 and 3domains), MHC-II RT1.B^(u) (all domains) and MHC-II RT1.D^(u) (alpha2and beta1 domains) as previously published (17-19) and manufactured byGL Biochem Ltd (Shangai, China). Lyophilized peptides were dissolved in0.4% sterile DMSO/sterile water and stored at −80° C. As controlpeptides, we used in vitro various allogeneic non activating peptides#7, 26 and 39 and in vivo, we used peptide #31.

Degenerated 9 to 15-mer overlapping peptides with 1 to 2 aa lagging weredesigned to cover the sequence of positively isolated 16-mer peptide andsynthesized by GL Biochem Ltd (Shangai, China).

All peptides were shown to be >95% homogeneous by analytical reversephase HPLC and aa sequences were confirmed. Peptides were diluted incomplete RMPI-1640 at a concentration of 120 μg/ml.

Cell Purification:

T cells were purified as previously described (6). Briefly, totalsplenocytes were depleted with a cocktail of anti-γδ T cells (V65),anti-CD45RA cells (OX33), anti-CD161 NK cells (3.2.3) and anti-CD11b/cmonocytes (OX42) using magnetic beads (Dynal). Enriched T cells werelabeled with anti-CD45RC-biotin (OX22) and Strepavidin-PE-Cy7,anti-CD8α-PE (OX8), anti-TCRαβ-Alexa 647 (R73) and anti-CD25-FITC (OX39)mAbs. CD8⁺CD45RC^(low) T cells and CD4⁺CD25⁻ T cells were sorted aftergating of TCRαβ⁺ cells with FACSAria (BD Biosciences, Mountain View,Calif.). Purity of sorted populations was greater than 99%.

Plasmacytoid dendritic cells (pDCs) were purified as previouslydescribed (6). Briefly, splenocytes recovered after collagenasedigestion were further purified by negative depletion with anti-TCR (R73and V65) T cells and anti-CD45RA (OX33) B cells mAbs. Enriched cellswere labeled with anti-CD45R-PE (His24), anti-CD4-APC (OX35),anti-TCR-FITC (R73) and anti-CD45RA-FITC (OX33). pDCs, defined as CD45Rand CD4 positive cells, were sorted after gating on FITC negative cells.

Mixed Lymphocyte Reaction:

For MLR coculture assays, pDCs from LEW.1A naive rats (1.25×10⁴ cells),syngeneic CD8⁺CD40Ig Tregs (5×10⁴ cells) and 120 μg/ml of allogeneicpeptides were plated in triplicate in 200 μl of complete RPMI-1640medium in round-bottom 96 wells plates for 6 days at 37° C., 5% CO2.pDCs were matured with 0.5 μM of CpG ODN 1826.

For direct MLR suppressive assays, sorted CFSE-labeled CD4⁺CD25⁻ T cellsfrom LEW.1A origin (5×10⁴ cells) and allogeneic pDCs isolated from donorLEW.1W animals (1.25×10⁴ cells) were plated in triplicate for 6 days ina final volume of 200 μl of completed RPMI-1640 medium in round-bottom96 wells plates with FACS-sorted freshly purified naive CD8⁺CD45RC^(low)Treg cells, peptide-expanded CD8⁺CD40Ig Treg cells (5×10⁴ cells), Du51tetramer⁺ or ⁻ CD8⁺CD40Ig Tregs. For indirect MLR suppressive assay,splenocytes isolated from donor LEW.1W animals were frozen-thawed toinduce apoptosis. Apoptotic cells were then cultured overnight with pDCsisolated from recipient LEW.1A animals (8:1 ratio) at 0.65×10⁶ pDCs/mL.Alloantigens-loaded pDCs were finally washed and plated as describedbefore for the direct MLR.

For anti-CD3/anti-CD28 stimulations, round-bottom 96 wells plates werecoated with anti-CD3 (1 μg/mL, BD Pharmingen) and anti-CD28 (10 μg/mL)mAbs for 1 hour at 37° C., 5% CO2, then washed and 5·10⁴ CD8⁺CD40Ig Tregcells were added to each well in 200 μl completed RPMI-1640 for 1, 2, 3and 6 days.

Proliferation of CFSE-labeled naive CD4⁺CD25⁻ T cells and phenotype ofCD8⁺CD45RC^(low) Tregs were analyzed by flow cytometry on a FACSCanto IIcytometer (BD Biosciences, Mountain View, Calif.) after gating onTCR⁺CD4⁺ cells or TCR⁺CD8⁺ cells among live cells (DAPI negative).

Extracellular and Intracellular Staining:

For extracellular staining, cells were stained with the following mAbs:anti-TCRαβ (R73, Alexa Fluor 647-conjugated), anti-CD8α (OX8,PE-Cy7-conjugated, ebiosciences), anti-CD4 (W3.25, PE-Cy7-conjugated),anti-CD45RC (OX22, FITC-conjugated), anti-CD28 (JJ319, biotin-labeled),anti-CD71 (OX26, biotin-labeled), anti-mouse Vb11 (KT11, biotin-labeled,AbD Serotec) anti-CD25 (OX39, biotin-labeled) and anti-MHC-II (OX6,biotin-labeled).

For intracellular staining, cells were then stained for Foxp3(biotin-labeled, ebiosciences) using BD cytofix/cytoperm kit (BDBiosciences) according to the manufacturer's instructions.

All biotinylated mAbs were visualized using Streptavidin-PerCP.Cy5.5 (BDBiosciences). A FACSCanto II cytofluorimeter (BD Biosciences, MountainView, Calif.) was used to measure fluorescence, and data were analyzedusing FlowJo software (Tree Star, Inc. USA, version 7.6.5). Cells werefirst gated by their morphology and dead cells excluded by selectingDAPI negative viable cells.

Cytokine Assays:

IFNγ, IL-10 were measured in coculture supernatants using ELISA from BDBiosciences OptEIA, IL-12 and TGFβ using ELISA from Invitrogen and R&DSystem respectively.

Production of Biotinylated RT1-A^(a)-Peptide Complexes:

Briefly, the heavy chain RT1-A^(a) and the β2microglobuline (β2m) werecloned in pET24 and produced in Escherichia coli BL21-DE3. Recombinantproteins were produced as inclusion bodies, dissolved in 8M urea andrefolded in vitro as previously described for human HLA-A2/peptidecomplexes (61). RT1-A^(a), β2m and peptide Du51 were refolded in 0.4ML-arginine, 0.1M Tris pH8, 2 mM EDTA, 5 mM reduced glutathione, and 0.5mM oxydated glutathione for 5 days at 4° C. The solution was thenconcentrated and the buffer changed on amicon membrane 10 Kd (Millipore,Bedford, Mass.). Folded MHC/peptide complexes were biotinylated with theBirA enzyme (Avidity, Denvers Colo.) for 5 h at 30° C. and desalted onHiprep 26/10 desalting column (GE Healthcare). MHC/peptide complexeswere then purified by anion exchange Q-Sepharose chromatography.Biotinylation was tested by tetramerization with streptavidin (SigmaAldrich) at a molar ratio of 4:1.

Tetramerization and Staining:

Tetramerization of RT1.A^(a)/Du51 was performed at room temperature byaddition of streptavidin-PE (Jackson ImmunoResearch) or streptavidin-APC(BD Biosciences) at a 4:1-molar ratio, in four equal aliquots added at15-min intervals. Likewise, the control tetramer RT1.A^(a)/MTF-E(ILFPSSERLISNR) was conjugated to streptavidin-BV421 (Biolegend) andrepresented 1.6+/−0.7% of non-specific staining among Du51-specificcells. These three reagents were mixed and added to plated cells at 10μg/mL for 1 hour at 4° C. Cells were further stained for CD8 and CD45RCand fluorescence was analyzed on a FACSCanto II cytometer (BDBiosciences, Mountain View, Calif.). Cells were first gated by theirmorphology and dead cells excluded by selecting DAPI negative cells.

Peptides Treatment In Vivo:

16-mer peptides were dissolved in 0.4% DMSO/PBS before injection. Forthe first protocol, single doses of peptide (500 μg/injection) wereinjected i.v at different time points before and after transplantationat day −6, −3, 0 +3 and +7 into grafted LEW.1A recipients. In the secondprotocol, mini osmotic pumps (ALZET, Cupertino, Calif., USA) wereimplanted intraperitoneally (i.p) in recipients and deliveredcontinuously either 20.83 or 41.66 μg/hour of 16-mer peptides for 14days. A first pump was implanted on day −7 before transplantation andwas replaced by a second one at day +7, allowing delivery of 14 or 28 mgof peptide per animal for 28 consecutive days. A depleting anti-CD8α mAb(OX8, IgG1, 3 mg/kg) or an anti-MHC class Ia and Ib mAb (OX18, 3 mg/kg)were injected i.p. twice a week from day −7 until rejection. Allograftswere monitored daily by palpation and allograft rejection was defined ascomplete cessation of palpable heart beat.

Adoptive Cell Transfer:

Cell transfers were performed by i.v. injection of purified sorted totalor Du51 tetramer⁻ CD8⁺CD40Ig Tregs into LEW.1A recipients sublethallyirradiated (4.5 Gy whole-body irradiation) on the day beforetransplantation. Total splenocytes (1.5·10⁸ cells) were adoptivelytransferred i.v. the day before heart transplantation into naive LEW-1Arecipients that had received 4.5 Gy of whole-body irradiation the sameday. Recipients received splenocytes from Du51-treated rats (defined as1^(st) spl-transferred) or from naive rats (naive spl-transferred) or nocells (untreated irradiated).

Morphometric Analysis of Cardiac Grafts:

The upper third of the graft was fixed in paraformaldehyde and embeddedin paraffin. Five μm coronal sections were stained withhematoxylin-eosine-safron. Tissues were analyzed by a pathologist (K.R.)blinded to the groups and chronic rejection was evaluated as previouslydescribed (63).

Donor Specific Alloantibody Detection:

Alloantibodies were analyzed by cytofluorimetry as described elsewhere(28). Briefly, after digestion by collagenase D and red blood celllysis, allogeneic spleen cells were incubated with diluted (1/8)heat-inactivated serum, and then with FITC-conjugated goat anti-rat IgGantibodies (H+L chain specific) (Jackson Laboratories), a mouse anti-ratIgG1 MAb (MCA 194, Serotec), IgG2a (MCA 278, Serotec) or IgG2b (MCA 195,Serotec). Antibody binding was revealed using FITC-coupled F(ab)′2 goatanti-mouse IgG (Jackson Laboratories). Cells were analyzed using a FACSCanto II cytofluorimeter (BD Biosciences, Mountain View, Calif.) and theresults were expressed as mean channel fluorescence for each serum.

Statistical Analysis:

For the peptide activation test, a non-parametric Wilcoxon signed-ranktest, comparing column median to a hypothetical value of 1.0, was done.Statistical significance for the TCR Vβ11 expression, phenotype ofactivated cells, cytokine expression, proliferation assay and tetramerstaining was evaluated by a two-tailed Mann Whitney t test. Graftsurvival was analyzed by Kaplan-Meier log-rank test. The Two-Way ANOVAtest and Bonferroni post-tests were used for donor-specific antibodyanalysis and splenocyte phenotypic characterization. Analyses were madewith GraphPad Prism 5.04 software (GraphPad, San Diego, Calif., USA).For the diversity analysis, Kruskal-Wallis and Dunn's multiplecomparison post-test were performed using GraphPad Prism 6.0c. A P-valueless than 0.05 was considered significant.

Results

CD8⁺CD40Ig Tregs Activation In Vitro.

In order to identify TCR recognition of allogeneic MHC/peptide complexesby CD8⁺CD40Ig Tregs and subsequent activation of their function, we hadto select a specific marker of activation allowing analysis by flowcytometry following exposure to antigenic stimulation. Therefore, wescreened molecules expressed at different time points by CD8⁺CD40IgTregs upon stimulation with polyclonal anti-CD3 and anti-CD28antibodies. Expression of molecules on freshly isolated CD8⁺CD40Ig Tregshas been previously assessed by Q RT-PCR (5) and demonstrated that amongthese molecules, CD25 and IFNγ were markers distinguishing CD8⁺CD40IgTregs from other cell populations. We analyzed by flow cytometry at day0, 1, 2, 3 and 6 their expression of CD71, CD25 and IFNγ.

We confirmed, at day 0, that CD8⁺CD40Ig Tregs expressed low levels ofCD71 (0.83±0.1%), CD25 (12.74±6.1%), and IFNγ (5.57±3.3%). Afterpolyclonal stimulation, CD71, CD25 and IFNγ expression increasedsignificantly from the first day and remained stable over time withrespectively 82±4.5%, 98.1±1.9% and 91.7±7% of positive cells at day 6.

In conclusion, we identified three markers of interest to CD8⁺CD40IgTregs with low basal expression and significant up-regulation uponstimulation. Since CD25 was the most and the earliest up-regulatedmarker and since it was a marker previously described by us and others(5, 12), we selected this marker to assess CD8⁺CD40Ig Treg activationfor the remaining aspects of this study.

CD8⁺CD40Ig Tregs Cells Recognized Two Donor MHC Class 11-DerivedPeptides.

In the rat MHC-mismatched heart allograft model, donors (RT1^(u)) andrecipients (RT1^(a)) are mismatched for all MHC molecules. We thereforealigned donor and recipient MHC I and II amino acids (aa) sequences anddesigned 82 overlapping 16-mer peptides matching the polymorphic domainsof donor MHC I and II molecules (17-19). Peptides were first groupedinto pools of 6 to 8 peptides (30 μg/ml of each peptide) and tested inan in vitro assay where immature or mature syngeneic recipient pDCs andsorted-CD8⁺CD40Ig Tregs from CD40Ig-treated long-term allograft bearingrecipients were cocultured for 3 or 6 days. With immature pDCs, weobserved no significant activation of CD8⁺CD40Ig Tregs at day 3 or day 6with any of the allogeneic pools of peptides. After stimulation withmature pDCs and pools of allogeneic peptides we observed at day 3 aslight upregulation of CD25 expression of a small population ofCD8⁺CD40Ig Tregs, and at day 6, a significant up-regulation of CD25expression following allogeneic stimulation. These results suggestedthat some allogeneic peptides were efficiently recognized by CD8⁺CD40IgTregs and that this recognition led to increased CD25 expression. Italso demonstrated that pDCs must be matured in our assay.

We next tested the stimulatory capacity of the 82 individualallopeptides in the presence of naive matured syngeneic pDCs andCD8⁺CD40Ig Tregs purified from long-term survivors in a 6 days culture(FIG. 1A). We observed that two peptides induced a highly significantupregulation of CD25 expression at the cell surface of CD8⁺CD40Ig Tregs:peptide #31 (called Bu31, 1.67±0.09 fold vs. no peptide, p<0.0001),whose sequence overlaps with peptide #32 sequence (p<0.05), and peptide#51 (called Du51, 2.07±0.18 fold vs. no peptide, p<0.001). Du51 induceda stronger upregulation of CD25 expression compared to Bu31, suggestingthat Du51 is the dominant peptide recognized by CD8⁺CD40Ig Tregs, whileBu31 is sub-dominant. These results demonstrated that antigen-specificCD8⁺CD40Ig Tregs mainly recognized two peptides, Bu31 (YLRYDSDVGEYRAVTE)and Du51 (NREEYARFDSDVGEYR), derived respectively from the 131 chain ofdonor MHC class II RT1.B^(u) and RT1.D^(u) molecules.

CD8⁺CD40Ig Tregs Recognized an Unusually Long Allogeneic 15-Mer Peptide.

To determine the sequence of the natural dominant donor peptiderecognized by antigen-specific CD8⁺CD40Ig Tregs, we used a library ofdegenerated peptides, ranging from 9-mer peptides with one aa lagging to15-mer peptides with two or more aa lagging derived from the dominant16-mer Du51 (labeled #51-1 to #51-18) (FIG. 1B and Table 1). Thislibrary's design was based on previous results and published reports(20-23) and tested in the same in vitro assay described above.Interestingly, random peptide libraries have established that rat MHCclass I RT1-A^(a) molecules showed a strong preference for 9 to 15 merpeptides bearing an arginine (R) at the C terminus (23).

TABLE 1 List of peptides used in the present study: Length SEQ NameSequence (aa) ID NO: Du51 NREEYARFDSDVGEYR 16  2 51-1 NREEYARFD  9 1151-2 REEYARFDS  9 12 51-3 EEYARFDSD  9 13 51-4 EYARFDSDV  9 14 51-5YARFDSDVG  9 15 51-6 ARFDSDVGE  9 16 51-7 RFDSDVGEY  9 17 51-8 FDSDVGEYR 9 18 51-9 YARFDSDVGE 10 19 51-10 EYARFDSDVG 10 20 51-11 EYARFDSDVGE 1121 51-12 YARFDSDVGEY 11 22 51-13 YARFDSDVGEYR 12 23 51-14 EYARFDSDVGEY12 24 51-15 EEYARFDSDVGE 12 25 51-16 EEYARFDSDVGEY 13 26 51-17EEYARFDSDVGEYR 14 27 51-18 REEYARFDSDVGEYR 15  1

None of the derivative 9-mer peptides #51-1 to #51-8 was able to induceactivation of CD8⁺CD40Ig Tregs equivalent to the one observed with the16-mer Du51. However, we were able to induce a much stronger andsignificant CD25 upregulation with a 15-mer derivative peptide (51-18)(2.04±0.3 fold vs. no peptide) missing the N-term asparagine (N).Contrary to other derivatives, CD25 upregulation induced by peptide#51-18 was not significantly different from that induced by Du51 (FIG.1B).

Altogether, these results showed that a dominant MHC class II-derived15-mer natural peptide (REEYARFDSDVGEYR) was presented to the CD8⁺CD40IgTregs and that such presentation induced activation of the specificcells.

Du51-Activated CD8⁺CD40Ig Treg Cells Displayed a Modified Phenotype andEfficiently Suppressed Antigen-Specific Activated T Cells.

The phenotype of Du51-activated CD8⁺CD40Ig Tregs was analyzed 6 daysafter stimulation. We previously demonstrated that CD8⁺CD40Ig Tregsacted through secretion of high levels of IFNγ, that in turn induced IDOexpression by DCs and graft ECs and this action was necessary fortolerance induction in vivo (5). According to these results, we observedthat stimulation of CD8⁺ Tregs by the peptide in the presence of pDCsled to significant increased expression of IFNγ, most likely byactivated CD8⁺CD40Ig Tregs. In the same culture supernatants, weobserved decreased IL-12 production, likely of pDC origin, but nomodification of IL-10 and TGFβ expression that could be produced by bothCD8⁺CD40Ig Tregs and pDCs. We also observed an upregulation of CD71,CD28 and MHC class II, but no modification of Foxp3 expression after 6days of peptide stimulation.

We previously demonstrated that CD8⁺CD40Ig Tregs, in the presence ofallogeneic pDCs or syngeneic pDC and a lysate of donor cells, couldsuppress the proliferation of syngeneic effector CD4⁺CD25⁻ T cells,showing that CD8⁺CD40Ig Tregs acted through the direct and indirectpathway of allorecognition, and that they are more efficient suppressorcells than naive CD8⁺CD45RC^(low) Tregs (6). Here, we investigatedwhether Du51-stimulated CD8⁺CD40Ig Tregs could efficiently suppresseffector T cell proliferation after 6 days coculture and thus maintainedtheir suppressor activity, compared to CD8⁺CD40Ig Tregs stimulated witha non-activating control peptide. We performed a MLR assay stimulatingCD8⁺CD40Ig Tregs for 6 days in the presence of syngeneic pDCs and Du51or non-activating peptide. Peptides-stimulated CD8⁺CD40Ig Tregs werethen sorted using a FACS Aria and added in a direct MLR assay of sortedallogeneic pDCs and syngeneic CFSE-labeled CD4⁺CD25⁻ effector T cells.We hypothesized that expanded-CD8⁺CD40Ig Tregs would exert bystanderregulation of the proliferation of effector CD4⁺CD25⁻ T cells stimulatedby the direct allorecognition pathway, that was measured 6 days later(24, 25). In the absence of Tregs, 80.5% of CD4⁺CD25⁻ effector T cellsproliferated. The addition of Du51-stimulated Tregs resulted in asignificant inhibition of the proliferation of effector T cells comparedto Tregs stimulated by a non-activating peptide. Thus, Du51antigen-specific CD8⁺CD40Ig Tregs maintained an efficient suppressiveactivity after in vitro activation and Tregs that remained unstimulatedfor 6 days lost their suppressive activity and started to die byneglect.

Identification of Du51-Specific CD8⁺CD45RC^(low) Tregs Using MHC Class ITetramer Revealed their Enrichment in CD40Ig-Treated Recipients.

An important challenge in transplantation remains the identification ofantigen-specific Tregs as they represent a more potent suppressivepopulation and very few natural epitopes have been identified so far. Inaddition, to date, in the rat and transplantation settings, no toolswere available to directly visualize and detect antigen-specific CD8⁺Treg populations. To that end, we generated a MHC class I tetramerRT1.A^(a)/Du51 that was labeled with phycoerythrin (PE) andallophycocyanin (APC) and stained specific populations in the spleen andgraft.

Cells were first stained with a mixture of PE-conjugated andAPC-conjugated RT1.A^(a)/Du51 tetramers, together with a controltetramer RT1.A^(a)/MTF-E labeled with BV421. This dual fluorochromestrategy, previously described for antigen-specific CD8⁺ effector Tcells, allows the discrimination of signal and noise staining, asspecific CD8⁺ Tregs will bind equally to each version of RT1.A^(a)/Du51tetramer whereas random elements will not (26). Among double positivestained events, non peptide-specific cells can be visualized andexcluded as they bind to a control tetramer bearing the same heavy chainRT1.A^(a) but with an irrelevant peptide associated. Cells weresecondary stained with CD8-PeCy7 and CD45RC-FITC to identify eitherCD8⁺CD45RC^(low) Tregs or CD8⁺CD45RC^(high) T cells. With such astrategy, we were able to identify 2.19±0.6% in the spleen and1.16±0.25% in the graft of Du51-specific cells among CD8⁺CD40Ig Tregs.In the naive splenic CD8⁺CD45RC^(low) Tregs population, we evaluated theprecursor frequency at 0.73±0.2%, demonstrating that even 120 daysfollowing transplantation and CD40Ig treatment, the frequency was stillincreased around 3 times and that in naive animals, we were able toidentify a pool of donor-specific Tregs. This difference was true whenlooking at the percentage and absolute number of tetramer-positiveCD8⁺CD40Ig Tregs or naive CD8⁺CD45RC^(low) T cells in the spleen(0.624±0.128 vs. 0.173±0.071 respectively for percentage and623800±127700 vs. 172600±70500 for absolute number in the spleen,p<0.05). There was also significantly more Du51-specific cells amongCD8⁺CD40Ig Tregs than among CD8⁺CD45RC^(high) T cells from naive orCD40Ig-treated animals, in terms of the percentage of positive cellsamong each population and percentage or absolute number in the totalspleen, and the percentage in the graft. There was no difference betweenCD8⁺CD45RC^(low) and CD8⁺CD45RC^(high) T cells from naive animals in thespleen. Interestingly, tetramer-positive cells were localized both inthe graft and the spleen within the CD8^(high) subset ofCD8⁺CD45RC^(low) Tregs.

Altogether, these results demonstrated that we were able to generate afunctional RT1A^(a)/Du51 tetramer to detect alloantigen-specific CD8⁺Tregs, a population that was significantly increased upontransplantation and CD40Ig treatment.

Superior Suppressive Capacity of Du51-Specific CD8⁺CD45RC^(low) TregsMediated by Direct and Indirect Pathways of Allorecognition andRequirement for In Vivo Tolerance Induction.

We previously demonstrated the suppressive capacity of Du51-stimulatedCD8⁺CD40Ig Tregs. In this experiment, we wanted to study the suppressivecapacity of freshly sorted RT1A^(a)/Du51 tetramer-specific CD8⁺CD40IgTregs (FIG. 2A). Naive CD8⁺CD45RC^(low) Tregs, total CD8⁺CD40Ig Tregs,RT1A^(a)/Du51 tetramer negative (tet⁻) and RT1A^(a)/Du51 tetramerpositive (tet⁺) CD8⁺CD40Ig Tregs were sorted and incubated for 6 dayswith allogeneic (direct pathway) or alloantigens-loaded syngeneic pDCs(indirect pathway) and naive CFSE-labeled syngeneic CD4⁺CD25⁻ T cells.In this assay, RT1A^(a)/Du51 tetramer-specific CD8⁺CD40Ig Tregs wereactivated by tetramer binding (data not shown and (27)). As previouslydescribed, total CD8⁺CD40Ig Tregs suppressed more efficiently theproliferation of effector CD4⁺CD25⁻ T cells induced by both direct andindirect pathways of alloantigen presentation than naiveCD8⁺CD45RC^(low) Tregs (FIG. 2A). Interestingly, we observed asignificant difference between the suppressive potential of tee vs. tet⁻CD8⁺CD40Ig Tregs mediated by the direct pathway of allorecognition, withthe tee CD8⁺CD40Ig Tregs being the most potent suppressor cell subset(FIG. 2A). Although not significant but with a statistical trend(p=0.0571), the same difference was obtained regarding the indirectalloantigen presentation pathway (FIG. 2A). Moreover, suppression ofCD4⁺CD25⁻ T cell proliferation was more effectively achieved byDu51-specific Tregs when induced by indirect, rather than direct,alloantigen presentation pathway. Interestingly, non-specific tet⁻CD8⁺CD40Ig Tregs tend to be less suppressive than total CD8⁺CD40IgTregs, highlighting the important contribution of the Du51-specific CD8⁺Tregs in the overall suppressive capacity of the total CD40Ig Tregspool. These results suggested that Du51 antigen-specific CD8⁺ Tregs arethe most efficient Treg subpopulation of the total CD8⁺CD40Ig Tregspool.

To study the in vivo relevance of the differential ex vivo suppressiveeffect observed for Du51-specific CD8⁺CD40Ig Tregs, we performedadoptive cell transfer experiments. Total CD8⁺CD40Ig or RT1A^(a)/Du51tetramer negative (tet⁻) CD8⁺CD40Ig Tregs, were sorted and adoptivelytransferred into naive grafted irradiated recipients (FIG. 2B). Unliketotal CD8⁺CD40Ig Tregs, tet⁻CD8⁺CD40Ig Tregs (that were depleted in Du51antigen-specific cells) were unable to inhibit allograft rejection,demonstrating the crucial role of the antigen-specific CD8⁺CD40Ig Tregsin the tolerogenic activity of the total CD8⁺CD40Ig Treg pool preventingallograft rejection and promoting the infectious tolerance.

Tolerance Induction by In Vivo Peptide Treatment Correlated withIncreased Proportion of CD8⁺CD45RC^(low) Tregs and Total Inhibition ofAnti-Donor Antibody Responses.

To further determine the potential of the immunodominant peptideidentified in the in vivo generation of CD8⁺CD45RC^(low) Tregs and inallograft survival, animals were separately treated using two differentprotocols of peptide administration with no other treatment. In thefirst one, animals received five intravenous (i.v) injections of 500 μgof peptide (FIG. 3A). We observed that injections of either controlpeptide or Du51 were not sufficient to induce a significant prolongationof allograft survival (respectively, 11 and 9.5 days, n=4) (FIG. 3A). Inthe second one, to improve the efficacy of the treatment, and becausesuch small peptides are rapidly eliminated from the recipient's body, wetested mini-osmotic pumps with a constant intra-peritoneal delivery of20.8 μg of peptide per hour for 28 days, starting day-7 beforetransplantation. Interestingly, this protocol allowed significantprolongation of allograft survival (p<0.01 compared to control peptideand no treatment) with 25% of indefinite allograft survival using Du51compared to control peptide (FIG. 3A). To prove that tolerance inducedby peptide infusion was dependent on CD8⁺ T cells and MHC class Ipresentation, we co-treated recipient with peptide infusion and either adepleting anti-CD8 mAb (OX8) or a blocking anti-MHC class I mAb (OX18)(FIG. 3A) as previously described (5). Administration of both antibodiescompletely abolished allograft survival, indicating that recognition ofMHC class I/antigen by CD8⁺ T cells was required in the establishment oftolerance obtained by peptide infusion.

Interestingly, increased dose of Du51 administered by osmotic pumpdelivering 41.6 μg of peptide per hour induced an indefinite allograftsurvival in 80% of the recipients of Lewis 1W (LEW.1W) donor hearts(FIG. 3A). However, Brown Norway (BN) third party grafts were promptlyrejected at day 7 after transplantation (FIG. 3A), demonstrating thatpeptide Du51 infusion induces donor specific tolerance mediated by CD8⁺Tregs.

Grafted hearts and spleens of rejecting or long-term survivingrecipients treated with peptide Du51 were analyzed for signs of chronicrejection, presence of anti-donor antibodies, proportion of total andtetramer-positive CD8⁺CD45RC^(low) Tregs, and in vitro suppressiontowards CD4⁺ effector T cells (FIG. 3B, C, D). Anatomopathologicanalysis of the graft of long-term recipients showed no signs of chronicrejection according to a previously established score (28). In addition,we observed a trend for an increase in the percentage and absolutenumber of total CD8⁺ T cells, in particular of CD8⁺CD45RC^(low) Tregs inthe spleen (FIG. 3C), as well as tetramer-positive Du51-specific Tregs(FIG. 3D) in long-term surviving peptide Du51-treated recipientscompared to untreated or Du51-treated recipients that rejected theirgraft early. These results suggest that antigen-specificCD8⁺CD45RC^(low) Tregs were induced/amplified by in vivo peptidetreatment, while other subsets were not, and may be responsible oftolerance induction. We confirmed that these peptide-induced activatedCD8⁺CD45RC^(low) Tregs displayed suppressive activity ex vivo since theyefficiently inhibit effector CD4⁺ T cell proliferation in the samemanner as freshly purified CD8⁺CD40Ig Tregs. Finally, we observed atotal inhibition of total IgG, IgG1, IgG2a and IgG2b alloantibodiesproduction in long-term surviving Du51-treated recipients compared tountreated rats or Du51-treated recipients that rejected their graftearly, that could correlate with the absence of chronic rejection (28)(FIG. 3B).

Infectious Tolerance Following Du51 Monotherapy.

To assess the dominant suppressive potential of induced regulatory cellsinvolved in the long-term allograft survival generated by 1 mg/day of invivo peptide Du51 treatment. To do so, we performed adoptive celltransfer experiments using splenocytes of long-surviving recipients intonaive grafted sublethaly irradiated recipients, as we have done before(5) (FIG. 4A). First adoptive transfer of 150·10⁶ splenocytes intosecondary naive grafted irradiated recipients resulted in significantprolongation of allograft survival of 50% of the recipients,demonstrating the induction of dominant regulatory cells capable toinhibit allograft rejection in newly grafted irradiated recipients. Weinvestigated the anatomopathological status of the graft of firstadoptively transferred long term splenocytes recipients and observed acomplete absence of vascular lesions and obstructions (i.e. no signs ofchronic rejection). Finally, we observed a total inhibition of totalIgG, IgG1 and IgG2b alloantibodies production in long-term survivingfirst adoptively transferred recipients compared to untreated rats thatdisplayed high alloantibodies production and comparable to naive rat,that could correlate with the absence of chronic rejection (28) (FIG.4B).

DISCUSSION

Our current knowledge on how Tregs recognize peptides and the role ofthis recognition is very limited and mostly based on either transgenicmouse models using CD4⁺ Treg's TCR gene transfer (8, 11) or on murineQa-1-restricted CD8⁺ Tregs involved in autoimmune disease and cancer andwhose Qa-1-peptide repertoire has been described in the last few years(29). However, these studies suggest that antigen-specific Tregs arecrucially influencing the outcome of long-term transplantation andcontribute to the establishment of tolerance (30). Recent studies havecharacterized peptides recognized by CD8⁺ Tregs during cancer (an hemeoxygenase-1-derived peptide), autoimmunity (Vβ-derived peptide) or evenpregnancy (minor antigen-derived peptide) but not during transplantation(2). Transplantation is a particular setting to identify antigenrecognized by Tregs as the presence of the graft is a continuous sourceof alloantigens and that is most certainly essential for the functionand maintenance of regulatory populations and thus the survival of thegrafted organ (31). The recognition of alloantigens in the context ofregulation has been shown by us and others to occur mainly by theindirect pathway of presentation, and in particular supported by pDCs(6, 30, 32).

In this report, we demonstrated for the first time that CD8⁺CD45RC^(low)Tregs, through the indirect pathway of presentation, can recognize onedominant allopeptide, named Du51, (and one sub-dominant) derived fromthe β1 domain of natural donor MHC class II molecules. These peptidesshare 80-90% homology with human HLA class II molecules and thus couldbe used to detect specific CD8⁺ Tregs in humans. By the use of a MHC-Ispecific tetramer, we showed that Du51-specific CD8⁺CD45RC^(low) Tregswere enriched in CD40Ig-treated long-term surviving recipients,expressed a biased restricted Vβ11 chain, displayed a strong suppressiveactivity ex-vivo and played a crucial role in tolerance induction uponadoptive transfer. Finally, peptide Du51 was shown to induceprolongation of allograft survival in vivo, inducing donor-specific CD8⁺Tregs.

Here, we described that the peptide Du51 displayed an unusual length of15 aa and that shorter peptides tested failed to induce significantrecognition by CD8⁺CD40Ig Tregs. Most of the literature has focused onshort peptides (8-10 aa) bound to MHC class I, although it is known that5-10% of peptides are longer peptides (more than 10 aa) that can bepresented by MHC class I molecules (33, 34). So far, such peptides havebeen identified for CTL models and are mostly derived from viralantigens. To our knowledge, we provide the first description of a 15 aapeptide that can be structurally recognized by CD8⁺ Tregs, as shown withour RT1A^(a)/Du51 tetramers. Recent studies suggested that these longpeptides drive a TCR recognition more focused on the peptide (35) andthat TCRs recognize MHC class I peptides of a preferential length (36).The rat MHC class I molecule RT1.A^(a) has been known to accommodateparticularly long peptides, with key-position residues as Gln, Met orLeu at P2, Phe at P3, Pro at P4 and Arg at the C terminus (21, 22).Speir et al has demonstrated previously in a model of maternallytransmitted minor histocompatibility antigen (MTF-E) of 13 residues thatimportant anchor residues (in particular arginine at position 13 (P13))allowed binding with considerable bulged conformation (37). We alsoobserved that Du51 displayed an Arg at the C-term end and thus couldhelp for RT1.A^(a) accommodation of the peptide. Interestingly, some ofthe peptides that were tested by us were also tested by Ballet et al. onCD4⁺ and CD8⁺ T cells isolated from rejected untreated animals(including dominant peptide Du51) in the same mismatched cardiacallograft model (LEW.1W into LEW.1A) as us (18). They found twoimmunodominant peptides, referred by us as peptide #47 and peptide #55,all derived from LEW.1W RT1.D^(u) molecules, involved in acute rejectionof grafts from unmodified LEW.1A recipients. Importantly, peptide Du51was not involved in acute rejection in their model and the twoimmunodominant peptides identified by Ballet et al. were not recognizedby our CD8⁺CD40Ig Tregs, suggesting that Tregs and non-Tregs did notrecognize the same antigens.

We were able to produce a RT1.A^(a)/Du51-tetramer, which in addition tobeing a valuable tool to track antigen-specific cells, can also be usedto determine TCR fine specificity and affinity. With this tetramer, weidentified in naive animals a pool of antigen-specific Treg precursorsof 0.73%, which was expanded around three times by 120 days followingtransplantation and CD40Ig treatment. This precursor frequencycorrelated with the observations of Leavenworth et al. made in a modelof arthritis in mice where they analyzed the occurrence ofQa-1-Hsp60_(p216) and Qa-1.R72A-Qdm tetramer-specific CD8⁺ Tregs anddescribed a naive frequency of respectively ˜1.65% and 0.46% of positiveCD8⁺ T cells for each (38). These observations suggest that precursorfrequency of antigen specific cells in a Treg population might be higherthan the frequency of a given antigen-reactive non-Treg T cells.

By the use of this MHC-I tetramer, we compared the suppressive capacityof Du51-specific and non-specific CD8⁺CD40Ig Tregs ex vivo in acoculture assay and in vivo through adoptive transfer of tetramer⁻CD8⁺CD40Ig Tregs (depleted in tetramer⁺ CD8⁺CD40Ig Tregs). Wedemonstrated ex vivo the superior suppressive potential of tetramer overtetramer⁻ CD8⁺CD40Ig Tregs, which significantly inhibit CD4⁺ effector Tcells proliferation stimulated by both the direct and indirect pathwaysof presentation, and most efficiently in the indirect allorecognitionsetting. These results are in agreement with several studiesdemonstrating the superior suppressor potential of Tregs of indirectspecificity in vitro (39, 40). In vivo, we observed a rapid allograftrejection in naive grafted irradiated recipients transferred withtetramer⁻ CD8⁺CD40Ig Tregs compared to recipients adoptively transferredwith total CD8⁺CD40Ig Tregs. In a similar manner, adoptive transfer oftet⁻CD8αα⁺ Tregs (depleted in tetramer Qa-1/Hsp60_(p216)-specific Tregs)could not prevent the development of autoimmune arthritis in a mousemodel (38). Also, Tsang et al. showed a crucial role of CD4⁺ Tregs ofindirect allospecificity in transplantation tolerance, demonstratingthat only adoptive transfer of TCR-transduced CD4⁺ Tregs of both directand indirect specificity, and not CD4⁺ Tregs of direct specificity only,can inhibit cardiac allograft rejection (11).

We have also analyzed different markers potentially expressed byantigen-specific regulatory T cells. Foxp3 does not seem to be aninteresting marker of CD8⁺CD40Ig Tregs unstimulated or stimulated withpeptide, although upon several rounds of strong anti-CD3/anti-CD28/IL-2stimulation, we were able to detect substantial levels of Foxp3 (datanot shown). We also found increased IFNγ production and decreased IL-12expression after peptide-specific stimulation. IFNγ was already shown byus as a crucial cytokine in our model (6).

Regarding the repertoire of induced CD8⁺CD40Ig Tregs, we previouslyshowed that these cells preferentially used a TCR that recombined theVβ11 chain and displayed a CDR3β of 9 aa in the spleen, suggesting theexpansion of an oligoclonal population of Tregs (5). However, sequencingof around 700 CDR3β across six long-surviving animals demonstrated thattotal Vβ11⁺CD8⁺CD40Ig Tregs displayed a relatively diverse repertoire inthe spleen. Nevertheless, a more frequent repeated sequence was found insome animals. Analysis of the TCR repertoire in the graft revealed thepredominance of two TCR-Vβ chains: Vβ11 (as in the spleen) and Vβ18.Unlike the spleen, sequencing revealed biased and restricted repertoiresfor both chains, with some shared clonotypes for the Vβ18 TCR, as in thedonor-specific blood transfusion model of induction of long-termallograft survival (41). However, the public sequence described byDouillard et al. was only found once in one animal of our study and didnot represent the Vβ18-repertoire in our model. One interestinghypothesis would be that CD8⁺CD40Ig Tregs bearing such biased andrestricted clonotypes are more potent suppressors and migrate early inthe graft to exert their inhibitory activity, and then stay localized inthe tolerated transplant where immune regulation is required (42). Incontrast, total CD8⁺CD40Ig Tregs that reside in the spleen but displayeda non (Vβ18) or less (Vβ11) restricted repertoire (and consequentlycloser to the naive Tregs' repertoire) potentially identify distinctregulatory populations (that possibly do not express the same chemokinereceptors) being recruited upon subsequent inflammation. Despite someshared clonotypes for both Vβ11 and Vβ18 (Suppl. Table 1), we could notfind public CDR3 sequences. Analyzing more precisely the repertoire ofantigen-specific CD8⁺CD40Ig Tregs in the spleen by focusing on tetramerDu51⁺ Tregs, we demonstrated reduced clonotypic diversity ofDu51-specific Vβ11⁺CD8⁺CD40Ig Tregs (compared to the total CD8⁺CD40IgTregs from naive and CD40Ig-treated spleen and similar to the graftCD8⁺CD40Ig Tregs), but not of Du51-specific Vβ18⁺CD8⁺CD40Ig Tregs. Thus,the Vβ18 chain usage might not be optimal for recognition of thisparticular antigen. However, even this analysis of the antigen-specificpopulation did not reveal public clonotypes for this Treg population.TCR sequences that are shared between individuals tend to be moreefficiently produced by a process of convergent recombination (43) andthus present at a higher frequency in the naive T cell repertoire(44-46). However, inter-individually shared TCR sequences are notnecessarily dominant in the immune response to antigen (47). Thisappears to be the case in our model, suggesting that this process may bemodified by either TCR expansion or TCRα-chain pairing. More recentpublications have revealed the important contribution of the alpha-chainin the specific pMHC recognition and how TCRαβ diversity should be takeninto consideration, as some specific TCR Vα pairings dictate and alterMHC restriction (48, 49). In the literature, a consensus on TCRdiversity of naive CD4⁺ Tregs was obtained stating that more is better.Authors demonstrated that a high TCR diversity ensures optimal Tregexpansion and function by increasing the probability of havingantigen-specific clones responding (13, 16, 50, 51). In our model, naiveCD8⁺ Tregs displayed a highly diverse repertoire that was remodeled andbiased by expansion of Du51-specific CD8⁺ Tregs after transplantationand CD40Ig treatment.

At last, from a therapeutic point of view, the identification of naturalpeptide recognized by regulatory T cells in transplantation is animportant goal as new strategies using amplified CD4⁺ Tregs are beingtested currently in human transplantation (30, 52). The difficulty isthat these human CD4⁺ Tregs amplified in a polyclonal way bynon-specific stimulus such as anti-CD3/CD28 antibodies are lessefficient than antigen-specific Tregs (2, 53), display limited expansioncapacity and are usually outgrown by conventional effector T cells (54).The use of specific antigen to expand Tregs in short-term culture wouldsurely improve clinical settings. As a proof of principle, weadministered the dominant peptide Du51 in naive grafted recipients,without immunosuppressive treatment, and observed significantprolongation of allograft survival. This is the first time this extendedsurvival can be obtained with allopeptide alone (i.e. withoutimmunosuppressive drugs) in rodents and clearly demonstrates both theefficiency of this peptide and the implication of the indirect pathwayof presentation in tolerance induction. Some studies had describedearlier immunomodulatory effects of HLA-derived peptides on alloimmuneresponses (55). Especially, an HLA-B7 derived-peptide, called Allotrap,was shown to prolong skin and heart allograft survival when associatedwith ciclosporin administration in mice and rat respectively (56, 57).Its effect was associated with modulation of heme-oxygenase 1 activity(58). In our model, administration of either anti-CD8 or anti-MHC-Iantibodies in combination with peptide Du51 completely abolishedallograft survival. Thus, the in vivo therapeutic effect obtained withpeptide Du51 infusion was directly linked to MHC class I presentationand CD8⁺ T cell induction. Moreover, the immunodominant peptide Du51induced a donor-specific inhibition of alloimmune responses as thirdparty grafts were promptly rejected. In addition, the peptideDu51-induced allograft survival was accompanied by a total inhibition ofanti-donor antibodies that is probably related to the occurrence ofantigen-specific CD8⁺ Tregs after infusion of the allopeptide. Thisresult is important as it shows the potency of peptide-induced Tregs toinhibit acute and chronic allograft rejection occurrence, and opens newpossibilities in human transplantation. In addition, such expandedhighly suppressive Tregs could provide us with more fundamentalinformation on new and poorly described genes overexpressed with suchconditions and that could be used as biomarkers (6). Finally, we couldassume that the presence of this CD8⁺CD45RC^(low) Treg population insome groups of patients could be associated with a better prognosisduring the course of diseases (59, 60).

In conclusion, our study indicates that MHC class II donor antigen canbe used to boost antigen-specific CD8⁺ Treg generation and/or function,and that in turn, these Tregs inhibit anti-donor immune responsesallowing the establishment of a true tolerance. We also demonstratedthat antigen-specific CD8⁺ Tregs' TCRs display a private and restrictedrepertoire that ensures efficient expansion and suppression ofalloreactive immune responses. Altogether, these results highlight theimportance of the TCR, of its interaction with MHC/peptide and open newpossibilities in the generation of this population that could betransferrable to human settings.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. An isolated peptide derived from a MHCclass II molecule, wherein the isolated peptide has a length of from 15to 40 amino acids, and wherein the isolated peptide has an amino acidsequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acidsequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or comprises a variant of SEQID NO:1 selected from the group consisting of: REEYARFDSDVGEFR (SEQ IDNO: 3), REEYVRFDSDVGEYR (SEQ ID NO: 4), QEEYARFDSDVGEYR (SEQ ID NO: 5),REEYARFDSDVGVYR (SEQ ID NO: 6), NREEYARFDSDVGEFR (SEQ ID NO: 7),NREEYVRFDSDVGEYR (SEQ ID NO: 8), NQEEYARFDSDVGEYR (SEQ ID NO: 9) andNREEYAREDSDVGVYR (SEQ ID NO: 10), with the caveat that the isolatedpeptide is not NREEYARFDSDVGEYR (SEQ ID NO: 2).
 2. The isolated peptideaccording to claim 1, wherein the isolated peptide is 16 amino acids inlength.
 3. A nucleic acid sequence encoding the peptide of claim
 1. 4. Avector comprising the nucleic acid sequence of claim
 3. 5. A host cellcomprising the vector of claim
 4. 6. A MHC/peptide multimer comprising apeptide sequence encoding multiple peptides, wherein at least oneencoded peptide is derived from a MHC class II molecule and has a lengthof from 15 to 25 amino acids, and wherein the at least one encodedpeptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) orcomprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), orcomprises a variant thereof, selected from the group consisting of:(SEQ ID NO: 2) NREEYARFDSDVGEYR, (SEQ ID NO: 3) REEYARFDSDVGEFR;(SEQ ID NO: 4) REEYVRFDSDVGEYR; (SEQ ID NO: 5) QEEYARFDSDVGEYR;(SEQ ID NO: 6) REEYARFDSDVGVYR; (SEQ ID NO: 7) NREEYARFDSDVGEFR;(SEQ ID NO: 8) NREEYVRFDSDVGEYR; (SEQ ID NO: 9) NQEEYARFDSDVGEYR; or(SEQ ID NO: 10) NREEYARFDSDVGVYR.


7. In vitro or ex vivo method for generating a population ofCD8+CD45RC^(low) Tregs, comprising a step of culturing a population ofCD8+ Tregs with a culture medium comprising i) an isolated peptidederived from a MHC class II molecule, wherein the isolated peptide has alength of from 15 to 25 amino acids, and wherein the isolated peptidehas an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprisesthe amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1) or ii) comprisesa variant of SEQ ID NO: 1 selected from the group consisting of:NREEYARFDSDVGEYR (SEQ ID NO: 2); REEYARFDSDVGEFR (SEQ ID NO: 3):REEYVRFDSDVGEYR (SEQ ID NO: 4); QEEYARFDSDVGEYR (SEQ ID NO: 5);REEYARFDSDVGVYR (SEQ ID NO: 6); NREEYARFDSDVGEYR (SEQ ID NO: 7);NREEYVRFDSDVGEYR (SEQ ID NO: 8); NQEEYARFDSDVGEYR (SEQ. ID NO: 9) andNREEYARFDSDVGVYR (SEQ ID NO: 10), wherein the step of culturing isperformed in the presence of a population of plasmacytoid dendriticcells.
 8. An in vitro or ex vivo method for generating a population ofCD8±CD45RC¹″ Tregs, comprising a step of culturing a population of CD8+Tregs with a culture medium comprising a MHC/peptide multimer comprisingi) a peptide derived from a MHC class II molecule, wherein the peptidehas a length of from 15 to 25 amino acids, and wherein the peptide hasan amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1), or ii) a variant ofSEQ ID NO:1 selected from the group consisting of: (SEQ ID NO: 2)NREEYARFDSDVGEYR, (SEQ ID NO: 3) REEYARFDSDVGEFR; (SEQ ID NO: 4)REEYVRFDSDVGEYR; (SEQ ID NO: 5) QEEYARFDSDVGEYR; (SEQ ID NO: 6)REEYARFDSDVGVYR; (SEQ ID NO: 7) NREEYARFDSDVGEFR; (SEQ ID NO: 8)NREEYVRFDSDVGEYR; (SEQ ID NO: 9) NQEEYARFDSDVGEYR; or (SEQ ID NO: 10)NREEYARFDSDVGVYR.


9. A method of inducing tolerance in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of i) a peptide derived from a MHC class II molecule, wherein thepeptide has a length of from 15 to 25 amino acids, and wherein thepeptide has an amino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) orcomprises the amino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1); orii) comprises a variant of SEQ ID NO:1 selected from the groupconsisting of: (SEQ ID NO: 2) NREEYARFDSDVGEYR, (SEQ ID NO: 3)REEYARFDSDVGEFR, (SEQ ID NO: 4) REEYVRFDSDVGEYR, (SEQ ID NO: 5)QEEYARFDSDVGEYR, (SEQ ID NO: 6) REEYARFDSDVGVYR, (SEQ ID NO: 7)NREEYARFDSDVGEFR, (SEQ ID NO: 8) NREEYVRFDSDVGEYR, (SEQ ID NO: 9)NQEEYARFDSDVGEYR and (SEQ ID NO: 10) NREEYARFDSDVGVYR; or

iii) comprises a multimer comprising the peptide or the variant of thepeptide.
 10. A method of preventing or reducing transplant rejection ina patient in need thereof in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of i) apeptide derived from a MHC class II molecule, wherein the peptide has alength of from 15 to 25 amino acids, and wherein the peptide has anamino acid sequence REEYARFDSDVGEYR (SEQ ID NO: 1) or comprises theamino acid sequence: REEYARFDSDVGEYR (SEQ ID NO: 1); ii) comprises avariant of SEQ ID NO:1 selected from the group consisting of:(SEQ ID NO: 2) NREEYARFDSDVGEYR, (SEQ ID NO: 3) REEYARFDSDVGEFR,(SEQ ID NO: 4) REEYVRFDSDVGEYR, (SEQ ID NO: 5) QEEYARFDSDVGEYR,(SEQ ID NO: 6) REEYARFDSDVGVYR, (SEQ ID NO: 7) NREEYARFDSDVGEFR,(SEQ ID NO: 8) NREEYVRFDSDVGEYR, (SEQ ID NO: 9) NQEEYARFDSDVGEYR and(SEQ ID NO: 10) NREEYARFDSDVGVYR; or

iii) a multimer comprising the peptide or the variant of the peptide.11. A pharmaceutical composition comprising a) a peptide of length offrom 15 to 40 amino acids which is derived from a MHC class II molecule,wherein the peptide comprises an amino acid sequence: REEYARFDSDVGEYR(SEQ ID NO: 1) or a variant thereof, selected from the group consistingof: (SEQ ID NO: 3) REEYARFDSDVGEFR, (SEQ ID NO: 4) REEYVRFDSDVGEYR,(SEQ ID NO: 5) QEEYARFDSDVGEYR, (SEQ ID NO: 6) REEYARFDSDVGVYR,(SEQ ID NO: 7) NREEYARFDSDVGEFR, (SEQ ID NO: 8) NREEYVRFDSDVGEYR,(SEQ ID NO: 9) NQEEYARFDSDVGEYR and (SEQ ID NO: 10) NREEYARFDSDVGVYR;

or b) a nucleic acid encoding the peptide; or c) an expression vectorcomprising the nucleic acid; or d) a host cell comprising the expressionvector; or e) a MHC/peptide multimer comprising the peptide; or f) anantigen-presenting cell comprising a complex comprising an MHC moleculeand the peptide; and a pharmaceutically acceptable carrier.
 12. An invitro method for determining whether a transplanted patient is tolerant,comprising a step of determining the presence of CD8+CD45RClow Tregs ina biological sample obtained from said transplanted patient, bycontacting said biological sample with a binding partner capable ofselectively interacting with CD8+CD45RClow Tregs, wherein the bindingpartner is a MHC/peptide multimer comprising i) a peptide derived from aMHC class II molecule, wherein the peptide has a length of from 15 to 25amino acids, and wherein the peptide has an amino acid sequenceREEYARFDSDVGEYR (SEQ ID NO: 1) or comprises the amino acid sequence:REEYARFDSDVGEYR (SEQ ID NO: 1), or ii) a variant of SEQ ID NO:1 selectedfrom the group consisting of: (SEQ ID NO: 2) NREEYARFDSDVGEYR,(SEQ ID NO: 3) REEYARFDSDVGEFR, (SEQ ID NO: 4) REEYVRFDSDVGEYR,(SEQ ID NO: 5) QEEYARFDSDVGEYR, (SEQ ID NO: 6) REEYARFDSDVGVYR,(SEQ ID NO: 7) NREEYARFDSDVGEFR, (SEQ ID NO: 8) NREEYVRFDSDVGEYR,(SEQ ID NO: 9) NQEEYARFDSDVGEYR and (SEQ ID NO: 10) NREEYARFDSDVGVYR;

wherein the presence of CD8+CD45RClow Tregs is indicative of tolerance.